Method for attaching one or more polynucleotide binding proteins to a target polynucleotide

ABSTRACT

The invention relates to new methods of attaching one or more polynucleotide binding proteins to a target polynucleotide. The invention also related to new methods of characterising target polynucleotides.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 15/113,207, filed Jul. 21, 2016, now U.S. Pat. No. 10,385,389), which is a national stage filing under U.S.C. § 371 of PCT International Application PCT/GB2015/050140, which has an international filing date of Jan. 22, 2015; is a continuation-in-part of PCT International Application PCT/GB2014/050175, which has an international filing date of Jan. 22, 2014; and claims foreign priority benefits under 35 U.S.C. § 119(a)-(d) or 35 U.S.C. § 365(b) of British application number 1406151.9, filed Apr. 4, 2014, British application number 1406155.0, filed Apr. 4, 2014, and British application number 1416197.0, filed Sep. 12, 2014, the contents of each of which are herein incorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to new methods of attaching one or more polynucleotide binding proteins to a target polynucleotide. The invention also relates to new methods of characterising target polynucleotides.

BACKGROUND OF THE INVENTION

There is currently a need for rapid and cheap polynucleotide (e.g. DNA or RNA) sequencing and identification technologies across a wide range of applications. Existing technologies are slow and expensive mainly because they rely on amplification techniques to produce large volumes of polynucleotide and require a high quantity of specialist fluorescent chemicals for signal detection.

Transmembrane pores (nanopores) have great potential as direct, electrical biosensors for polymers and a variety of small molecules. In particular, recent focus has been given to nanopores as a potential DNA sequencing technology.

When a potential is applied across a nanopore, there is a change in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time. Nanopore detection of the nucleotide gives a current change of known signature and duration. In the strand sequencing method, a single polynucleotide strand is passed through the pore and the identities of the nucleotides are derived. Strand sequencing can involve the use of a polynucleotide binding protein to control the movement of the polynucleotide through the pore.

SUMMARY OF THE INVENTION

The inventors have surprisingly demonstrated that it is possible to pre-load one or more polynucleotide binding proteins onto one or more loading moieties and then attach the one or more loading moieties to the target polynucleotide. It is surprising that the one or more polynucleotide binding proteins do not sterically hinder the attachment of the one or more loading moieties to the polynucleotide. It is also surprising that the attachment process does not affect the one or more polynucleotide binding proteins and that they retain their function and ability to bind to the one or more loading moieties after they are attached to the target polynucleotide.

Accordingly, the invention provides a method for attaching one or more polynucleotide binding proteins to a target polynucleotide, comprising:

(a) providing the one or more polynucleotide binding proteins bound to one or more loading moieties; and

(b) attaching the one or more loading moieties to the target polynucleotide.

The invention also provides method of characterising a target polynucleotide, comprising:

(a) carrying out a method of the invention;

(b) contacting the target polynucleotide having one or more polynucleotide binding proteins attached provided in step (a) with a transmembrane pore such that the one or more polynucleotide binding proteins control the movement of the polynucleotide with respect to the pore; and

(c) taking one or more measurements as the polynucleotide moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the polynucleotide and thereby characterising the target polynucleotide.

The invention also provides a method of preparing a target polynucleotide for characterisation, comprising:

(a) carrying out a method of the invention wherein the one or more polynucleotide binding proteins comprise one or more polymerases; and

(b) allowing the one or more polymerases attached to the target polynucleotide provided in step (a) to form one or more polynucleotides using the target polynucleotide as a template and thereby preparing the target polynucleotide for characterisation.

The invention also provides a method of characterising a target polynucleotide, comprising:

(a) carrying out a polymerase-based method of the invention;

(b) contacting the target polynucleotide and the one or more polynucleotides produced in step (a) with a transmembrane pore such that the target polynucleotide and the one or more polynucleotides move with respect to the pore; and

(c) taking one or more measurements as the target polynucleotide and the one or more polynucleotides move with respect to the pore wherein the measurements are indicative of one or more characteristics of the polynucleotides and thereby characterising the target polynucleotide.

The invention also provides:

-   -   a target polynucleotide modified using a method of the         invention;     -   a loading moiety having one or more bound polynucleotide binding         proteins; and     -   a kit for attaching one or more polynucleotide binding proteins         to a target polynucleotide, comprising (a) the one or more         polynucleotide binding proteins bound to one or more loading         moieties and (b) a ligase.

DESCRIPTION OF THE FIGURES

FIG. 1 shows an example FPLC trace after purification of the pre-bound TrwC Cba-L376C/Q594A/K762C to the DNA Hairpin-adapter using an 8 mL POROS HQ-10 column.

FIG. 2 shows an example FPLC trace after purification of the pre-bound TrwC Cba-L376C/Q594A/K762C to the DNA Hairpin-adapter using a 5 mL Histrap HP column.

FIG. 3 shows the TBE (native) PAGE gel analysis (the DNA calibration band corresponds to the DNA concentration shown at the top of the gel) column 1 shows the TrwC Cba-L376C/Q594A/K762C bound to the DNA Hairpin-adapter in buffer only (shown as band X), column 2 shows the TrwC Cba-L376C/Q594A/K762C bound to the DNA Hairpin-adapter after the TRIS buffer was added, column 3 shows the TrwC Cba-L376C/Q594A/K762C bound to the DNA Hairpin-adapter after the addition of Bismaleimideoethane, columns 4-6 show varying dilutions of TrwC Cba-L376C/Q594A/K762C bound to the DNA Hairpin-adapter after both FPLC purifications. Band X corresponded to enzyme bound to DNA. Band Y corresponded to DNA with no enzyme bound.

FIG. 4 shows the SDS PAGE gel analysis (the DNA calibration band corresponds to the DNA concentration shown at the top of the gel) column 1 shows the TrwC Cba-L376C/Q594A/K762C bound to the DNA Hairpin-adapter in buffer only (shown as band X), column 2 shows the TrwC Cba-L376C/Q594A/K762C bound to the DNA Hairpin-adapter after the TRIS buffer was added, column 3 shows the TrwC Cba-L376C/Q594A/K762C bound to the DNA Hairpin-adapter after the addition of Bismaleimideoethane, columns 4-6 show varying dilutions of TrwC Cba-L376C/Q594A/K762C bound to the DNA Hairpin-adapter after both FPLC purifications. Band Y corresponded to enzyme not closed on DNA. Band X corresponded to enzyme bound onto DNA by the Bismaleimideoethane linker.

FIG. 5 shows the TBE (native) PAGE gel analysis (the DNA calibration band corresponds to the DNA concentration shown at the top of the gel) column 1 shows the T4 Dda-E94C/C109A/C136A/A360C bound to the DNA hairpin-adapter before TMAD was added. Column 2 shows the T4 Dda-E94C/C109A/C136A/A360C bound to the DNA hairpin-adapter after the KCl and ATP were added, column 3 shows the T4 Dda-E94C/C109A/C136A/A360C bound to the DNA hairpin-adapter after the SPRI purification. Band 2:1 shows two enzymes bound, 1:1 shows one enzyme bound. The DNA band corresponded to DNA alone.

FIG. 6 shows an example current trace of when helicases T4 Dda-E94C/C109A/C136A/A360C and TrwC Cba-L376C/Q594A/K762C controlled the translocation of the DNA construct (see above current trace) through an MspA nanopore. The x-axis corresponds to the time (s) and the y-axis corresponds to the current (pA). The trace showed a single DNA strand moving through the nanopore under the control of the two helicases, the labelled regions 1 and 2 corresponded to the translocation of region 1 and 2 of the DNA construct. The trace shows a current trace observed when construct Y was translocated through the pore under the control of both T4 Dda-E94C/C109A/C136A/A360C and TrwC Cba-L376C/Q594A/K762C helicases. The arrow labelled 3 shows a spike in current as the spacers in the hairpin adapter translocated through the nanopore. The hairpin adapter and Y adaptor spacers are shown as an x in the DNA construct picture above the current trace.

FIGS. 7A-7B show an Agilent Bioanalyser trace illustrating that the enzyme was pre-bound to the MuA Y-adapter.

FIGS. 8A-8B show an Agilent Bioanalyser trace illustrating that when the enzyme was pre-bound to the MuA adapter no adverse effect was seen on the tagmentation of the target DNA.

FIG. 9 shows an example current trace of when a helicase controlled the movement of DNA through a nanopore that was prepared using DNA produced by MuA tagmentation.

FIGS. 10A-10B show the A-piece (see FIG. 10A) and the END-piece (see FIG. 10B) loading moieties which have E1 (A-piece) and E2 (END-piece) pre-bound. These two loading moieties are ligated to genomic DNA in Example 6. The A-piece has a region of 39 SpC3 spacers labelled 1. SEQ ID NO: 32 corresponded to the region labelled 2, this was the region to which E1 bound. The regions labelled 3 corresponded to four iSp18 spacers. The region labelled 4 corresponded to SEQ ID NO: 33. The regions labelled 5 corresponded to SEQ ID NO: 34. The regions labelled 6 corresponded to one iBNA-meC, two iBNA-A and two iBNA-meC. The regions labelled 7 corresponded to SEQ ID NO: 35. The region labelled 8 in the END-piece corresponded to SEQ ID NO: 36. The region labelled 9 corresponded to SEQ ID NO: 37. The region labelled 11 corresponded to SEQ ID NO: 38.

FIG. 11 shows an example plot of when helicases T4 Dda-(H82R/E94C/A360C) (SEQ ID NO: 24 with mutations H82R/E94C/A360C, E1) and T4 Dda-E94C/C109A/C136A/A360C (SEQ ID NO: 24 with mutations E94C/C109A/C136A/A360C, E2) controlled the translocation of the DNA construct in sample 4 (shown at the top of the figure) through an MspA nanopore. The x-axis corresponds to the movement index and the y-axis corresponds to the current (pA). For each DNA strand which moved through the pore the current was measured as a function of time. The moving DNA resulted in stepwise changes in the measured current levels. The observed current levels were fitted to obtain a mean current for each step and assigned an incrementing movement index point. The mean current against movement index therefore closely approximated the original current signal and was used to characterise the translocated DNA. The plot showed a single DNA strand moving through the nanopore under the control of the two helicases, the labelled regions 1 and 2 corresponded to the translocation of region 1 and 2 of the DNA construct. The trace shows the movement index observed when the construct was translocated through the pore under the control of both T4 Dda-(H82R/E94C/A360C) and T4 Dda-E94C/C109A/C136A/A360C helicases. The arrow labelled 3 shows a spike in current as the spacers in the hairpin adapter translocated through the nanopore. The hairpin adapter and Y adaptor spacers are shown as an x in the DNA construct picture above the trace.

FIGS. 12A-12C show the helicase-leader complex (see FIG. 12A), the polymerase strand complex (see FIG. 12B) and the final loading moiety the helicase/polymerase leader complex which had both a polymerase (labelled X1, Phi29-A411C/Q560C (SEQ ID NO: 9 with mutations A411C/Q560C)) and a helicase (labelled Y1, and T4 Dda-E94C/C109A/C136A/A360C (SEQ ID NO: 24 with mutations E94C/C109A/C136A/A360C)) pre-bound. This final loading moiety was ligated to a 3.6 kb DNA strand (SEQ ID NO: 46) in Example 7. The regions labelled 1 corresponded to 30 SpC3 spacers. SEQ ID NO: 27 corresponded to the regions labelled 2, this was the region to which T4 Dda-E94C/C109A/C136A/A360C bound. The regions labelled 3 corresponded to four iSp18 spacers. The regions labelled 4 corresponded to SEQ ID NO: 28. The regions labelled 5 corresponded to SEQ ID NO: 43. The regions labelled 6 corresponded to SEQ ID NO: 44 which was attached at its 3′ end to four iSpC3 spacers which were attached at the opposite end to the 5′ end of SEQ ID NO: 45. Phi29-A411C/Q560C bound to region 6.

FIG. 13 shows the DNA construct which was produced after the ligation step and before the polymerisation step in Example 7. The region labelled 1 corresponded to 30 SpC3 spacers. SEQ ID NO: 27 corresponded to the region labelled 2, this was the region to which T4 Dda-E94C/C109A/C136A/A360C bound. The regions labelled 3 corresponded to four iSp18 spacers. The regions labelled 4 corresponded to SEQ ID NO: 28. The regions labelled 5 corresponded to SEQ ID NO: 43. The regions labelled 6 corresponded to SEQ ID NO: 44 which was attached at its 3′ end to four iSpC3 spacers which were attached at the opposite end to the 5′ end of SEQ ID NO: 45. Phi29-A411C/Q560C bound to region 6. Region 7 corresponded to SEQ ID NO: 46. Region 8 corresponded to SEQ ID NO: 47. Region 9 corresponded to SEQ ID NO: 31.

FIGS. 14A-14B show an example plot of when the helicase T4 Dda-E94C/C109A/C136A/A360C (labelled Y1) controlled the translocation of the DNA construct in sample 5 (shown at the top of the figure) through an MspA nanopore. The x-axis corresponds to the movement index and the y-axis corresponds to the current (pA). For each DNA strand which moved through the pore the current was measured as a function of time. The moving DNA resulted in stepwise changes in the measured current levels. The observed current levels were fitted to obtain a mean current for each step and assigned an incrementing movement index point. The mean current against movement index therefore closely approximated the original current signal and was used to characterise the translocated DNA. The plot showed a single DNA strand moving through the nanopore under the control of the helicase, the labelled regions 1 and 2 corresponded to the translocation of region 1 and 2 of the original 3.6 kB DNA construct (SEQ ID NO: 46). The labelled regions 4 and 5 corresponded to the complementary strands to regions 1 and 2 which were produced by polymerisation using Phi29-A411C/Q560C. The trace shows the movement index observed when the construct was translocated through the pore under the control of T4 Dda-E94C/C109A/C136A/A360C. The arrow labelled 3 shows a spike in current as the spacers in the hairpin of the final construct (shown as x and labelled 3 in the top construct diagram of FIG. 14A) translocated through the nanopore. The hairpin adapter and Y adaptor spacers are shown as an x in the DNA construct picture above the plot.

FIG. 15 shows a 4-20% TBE PAGE which was run at 200V for 60 minutes and then stained using SYBR. Each sample run on the gel was 400 nM (5 μL). Lane 1 corresponded to a 100 bp ladder (the number of base pairs that the bands correspond to is shown along the side of the gel). Lane 2 corresponded to helicase leader complex shown in FIG. 12A, without the helicase bound. Lane 3 corresponded to the helicase leader complex shown in FIG. 12A with the helicase bound. Lane 4 corresponded to the polymerase strand complex shown in FIG. 12B without the polymerase bound. Lane 5 corresponded to the polymerase strand complex shown in FIG. 12B with the polymerase bound. Band A corresponded to SEQ ID NO: 43. Band B corresponded to DNA strand X=30 iSpC3 spacers attached at the 3′ end to the 5′ end SEQ ID NO: 27 which is attached at the 3′ end to 4 iSp18 spacers which are attached at the opposite end to the 5′ end of SEQ ID NO: 28. Band C corresponded to the helicase (T4 Dda-E94C/C109A/C136A/A360C) bound to DNA strand X. Band D corresponded to the polymerase strand complex without the enzyme bound (SEQ ID NO: 44 which was attached at its 3′ end to four iSpC3 spacers which were attached at the opposite end to the 5′ end of SEQ ID NO: 45). Band E corresponded to Phi29-A411C/Q560C bound to the polymerase strand complex.

FIGS. 16A-16B show an example of a method for ligation of adapters and polymerisation of a double-stranded target polynucleotide without pre-binding the polymerase (labelled x) to a loading moiety prior to ligation (FIG. 16A) and an example of the method of the invention of ligation of a loading moiety with both a pre-bound helicase (labelled y) and a pre-bound polymerase (labelled x) and then polymerisation of the double-stranded target polynucleotide (FIG. 16B). Step 1A shows ligation of adapters to either end of the double-stranded target polynucleotide. Step 2A shows binding of a polymerase (labelled x). Step 3A shows polymerisation of the double stranded target polynucleotide, the polynucleotide formed using the target as a template is shown as a dotted line. Step 4A shows end repair of the new double-stranded construct, A-tailing of the construct and ligation of a loading moiety with a pre-bound helicase (labelled y). Step 1B shows ligation of a loading moiety which contained both a pre-bound helicase (labelled y) and a pre-bound polymerase (labelled x). Step 2B shows polymerisation of the double-stranded target polynucleotide. No further steps were required in FIG. 16B, therefore, this process involved significantly fewer steps than the process shown in FIG. 16A.

FIG. 17 shows a loading moiety, comprising a pre-bound polymerase (labelled x) and a pre-bound helicase (labelled y), which is then ligated (step 1) to the two strands of the double stranded target polynucleotide (one labelled T1 for template and the other labelled C1 for complement) which is linked at one end by a bridging moiety adaptor (labelled z). In the loading moiety the helicase is bound to the opposite strand from the strand to which the polymerase is bound. The strand to which the polymerase is bound contains a 3′ hairpin loop (labelled v). In this embodiment, the polymerase will produce a double-stranded construct (step 2) in which the two strands of the construct are linked at one end by a bridging moiety (labelled v) and each strand of the construct comprises one strand of the target polynucleotide (shown as a solid line and labelled T1 and C1) and a complementary polynucleotide formed by the polymerases (shown as a dotted line and labelled T2 and C2).

FIG. 18 shows a loading moiety comprising a pre-bound polymerase (labelled x) and a pre-bound helicase (labelled y) which is then ligated (step 1) to each end of the two strands of the double stranded target polynucleotide, which are not linked at either end by a bridging moiety. In the loading moiety the helicase is bound to the opposite strand from the strand to which the polymerase is bound. The strand to which the polymerase is bound contains a 3′ hairpin loop (labelled v). In this embodiment, the polymerases will produce two double-stranded constructs (step 2) in which the two strands of each construct are linked at one end by a bridging moiety (labelled v1 or v2) and each construct comprises one strand of the target polynucleotide (shown as a solid line) and a complementary polynucleotide formed by the polymerases (shown as a dotted line).

FIG. 19 shows a loading moiety (bridging moiety adaptor labelled v and hydridized leader labelled w) comprising a pre-bound polymerase (labelled x) and a pre-bound helicase (labelled y), which is then ligated (step 1) to each end of the double stranded target polynucleotide so that they are linked at both ends by a loading moiety (labelled w and v as described previously) to form a circular construct. In this example the bridging moiety adaptor comprises a polynucleotide leader (labelled w) which has a helicase (labelled y) pre-bound to it, which is hybridised to the bridging moiety adaptor (labelled v). The double stranded section formed by the hybridisation of this polynucleotide (labelled w) to the bridging adaptor moiety forms a primer site for binding the polymerase. When polymerase extension is initiated (step 2) (eg. by the addition of nucleotides and cofactors) two constructs will be created with DNA that is a complementary copy of the original target DNA, and will contain multiple copied sections of T1 and C1 dependent on how far the polymerase proceeds around the circular construct.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encoding the MS-B1 mutant MspA monomer. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K.

SEQ ID NO: 2 shows the amino acid sequence of the mature form of the MS-B1 mutant of the MspA monomer. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K.

SEQ ID NO: 3 shows the polynucleotide sequence encoding one monomer of α-hemolysin-E111N/K147N (α-HL-NN; Stoddart et al., PNAS, 2009; 106(19): 7702-7707).

SEQ ID NO: 4 shows the amino acid sequence of one monomer of α-HL-NN.

SEQ ID NOs: 5 to 7 show the amino acid sequences of MspB, C and D.

SEQ ID NO: 8 shows the polynucleotide sequence encoding the Phi29 DNA polymerase. SEQ ID NO: 9 shows the amino acid sequence of the Phi29 DNA polymerase.

SEQ ID NO: 10 shows the codon optimised polynucleotide sequence derived from the sbcB gene from E. coli. It encodes the exonuclease I enzyme (EcoExo I) from E. coli. SEQ ID NO: 11 shows the amino acid sequence of exonuclease I enzyme (EcoExo I) from E. coli.

SEQ ID NO: 12 shows the codon optimised polynucleotide sequence derived from the xthA gene from E. coli. It encodes the exonuclease III enzyme from E. coli.

SEQ ID NO: 13 shows the amino acid sequence of the exonuclease III enzyme from E. coli. This enzyme performs distributive digestion of 5′ monophosphate nucleosides from one strand of double stranded DNA (dsDNA) in a 3′-5′ direction. Enzyme initiation on a strand requires a 5′ overhang of approximately 4 nucleotides.

SEQ ID NO: 14 shows the codon optimised polynucleotide sequence derived from the recJ gene from T. thermophilus. It encodes the RecJ enzyme from T. thermophilus (TthRecJ-cd).

SEQ ID NO: 15 shows the amino acid sequence of the RecJ enzyme from T. thermophilus (TthRecJ-cd). This enzyme performs processive digestion of 5′ monophosphate nucleosides from ssDNA in a 5′-3′ direction. Enzyme initiation on a strand requires at least 4 nucleotides.

SEQ ID NO: 16 shows the codon optimised polynucleotide sequence derived from the bacteriophage lambda exo (redX) gene. It encodes the bacteriophage lambda exonuclease.

SEQ ID NO: 17 shows the amino acid sequence of the bacteriophage lambda exonuclease. The sequence is one of three identical subunits that assemble into a trimer. The enzyme performs highly processive digestion of nucleotides from one strand of dsDNA, in a 5′-3′direction (http://www.neb.com/nebecomm/products/productM0262.asp). Enzyme initiation on a strand preferentially requires a 5′ overhang of approximately 4 nucleotides with a 5′ phosphate.

SEQ ID NO: 18 shows the amino acid sequence of He1308 Mbu.

SEQ ID NO: 19 shows the amino acid sequence of He1308 Csy.

SEQ ID NO: 20 shows the amino acid sequence of He1308 Tga.

SEQ ID NO: 21 shows the amino acid sequence of He1308 Mhu.

SEQ ID NO: 22 shows the amino acid sequence of TraI Eco.

SEQ ID NO: 23 shows the amino acid sequence of XPD Mbu.

SEQ ID NO: 24 shows the amino acid sequence of Dda 1993.

SEQ ID NO: 25 shows the amino acid sequence of Trwc Cba.

SEQ ID NO: 26 shows a polynucleotide sequence used in Example 1. This sequence has a 5′ phosphate.

SEQ ID NO: 27 shows a polynucleotide sequence used in Example 2 and 7.

SEQ ID NO: 28 shows a polynucleotide sequence used in Example 2 and 7.

SEQ ID NO: 29 shows a polynucleotide sequence used in Example 1. This sequence has a 5′ phosphate.

SEQ ID NO: 30 shows a polynucleotide sequence used in Example 1.

SEQ ID NO: 31 shows a polynucleotide sequence used in Example 7. This sequence has a 5′ cholesterol TEG.

SEQ ID NO: 32 shows a polynucleotide sequence used in Example 6. Attached to the 5′ end of SEQ ID NO: 32 is 39 SpC3 spacers. Attached to the 3′ end of SEQ ID NO: 32 is four iSP18 spacers which are attached at the opposite end to the 5′ end of SEQ ID NO: 33.

SEQ ID NO: 33 shows a polynucleotide sequence used in Example 6. Attached to the 5′ end of SEQ ID NO: 33 is four iSP18 spacers which are attached at the opposite end to the 3′ end of SEQ ID NO: 32.

SEQ ID NO: 34 shows a polynucleotide sequence used in Example 6. Attached to the 3′ end is one iBNA-meC, two iBNA-A and two iBNA-meC which are attached at the opposite end to three iSp18 spacers which are attached to 5′ end of SEQ ID NO: 35.

SEQ ID NO: 35 shows a polynucleotide sequence used in Example 6. Attached to the 5′ end is three iSp18 spacers which are attached at the opposite end to two iBNA-meC, two iBNA-A and one iBNA-meC which is attached at the opposite end to the 3′ end of SEQ ID NO: 34.

SEQ ID NO: 36 shows a polynucleotide sequence used in Example 6. Attached to the 5′ end of SEQ ID NO: 36 is a phosphate. Attached to the 3′ end of SEQ ID NO: 36 is four iSp18 spacers which are attached to the opposite end to the 5′ end of SEQ ID NO 37.

SEQ ID NO: 37 shows a polynucleotide sequence used in Example 6. Attached to the 5′ end of SEQ ID NO: 37 is four iSp18 spacers which are attached to the opposite end to the 3′ end of SEQ ID NO: 36.

SEQ ID NO: 38 shows a polynucleotide sequence used in Example 6.

SEQ ID NO: 39 shows a polynucleotide sequence used in Example 6. Attached to the 5′ end of SEQ ID NO: 39 is a phosphate. Attached to the 3′ end of SEQ ID NO: 39 is four iSpC3 spacers which are attached at the opposite end to the 5′ end of SEQ ID NO: 40.

SEQ ID NO: 40 shows a polynucleotide sequence used in Example 6. Attached to the 5′ end of SEQ ID NO: 40 is four iSpC3 which are attached at the opposite end to the 3′ end of SEQ ID NO: 39.

SEQ ID NO: 41 shows a polynucleotide sequence used in Example 6. SEQ ID NO: 41 is complementary to SEQ ID NO: 42.

SEQ ID NO: 42 shows a polynucleotide sequence used in Example 6. SEQ ID NO: 42 is complementary to SEQ ID NO: 41.

SEQ ID NO: 43 shows a polynucleotide sequence used in Example 7.

SEQ ID NO: 44 shows a polynucleotide sequence used in Example 7. This sequence has a 5′ phosphate. The 3′ end of SEQ ID NO: 44 is attached to four iSpC3 spacers which are attached at the opposite end to the 5′ end of SEQ ID NO: 45.

SEQ ID NO: 45 shows a polynucleotide sequence used in Example 7. This sequence is attached at its 5′ end to four iSpC3 spacers which are attached at the opposite end to the 3′ end of SEQ ID NO: 44.

SEQ ID NO: 46 shows a polynucleotide sequence used in Example 7.

SEQ ID NO: 47 shows a polynucleotide sequence used in Example 7. This sequence has a 5′ phosphate and a thymine based phosphorothioate base at the 3′ end of the sequence.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes two or more polynucleotides, reference to “an enzyme” includes two or more enzymes, reference to “a helicase” includes two or more helicases, reference to “a molecular brake” refers to two or more molecular brakes, reference to “a transmembrane pore” includes two or more pores and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Method of the Invention

The invention provides a method of attaching one or more polynucleotide binding proteins to a target polynucleotide. The one or more polynucleotide binding proteins are provided bound to (or attached to) one or more loading moieties. The one or more loading moieties are attached to the target polynucleotide. This attaches the one or more polynucleotide binding proteins to the target polynucleotide. Once the one or more polynucleotide binding proteins have been attached in this way, they can be used to control the movement of the target polynucleotide through a transmembrane pore or form one or more polynucleotides using the target polynucleotide as a template (i.e. modify the target polynucleotide). This allows the target polynucleotide to be characterised as discussed in more detail below.

The invention has various advantages.

-   i. Pre-loading the polynucleotide binding proteins on the loading     moieties speeds up the sample preparation process and means fewer     tubes are used. -   ii. As close to 100% as possible of the instances of the resulting     target polynucleotide have polynucleotide binding proteins attached.     This can improved yields of subsequent requisite processes. -   iii. Customer error is reduced if they do not load the one or more     polynucleotide binding proteins onto the one or more loading     moieties. -   iv. No excess polynucleotide binding protein (which could block the     pore or have some other unwanted activity) remains after attachment. -   v. Stability of the one or more polynucleotide binding proteins is     improved. A protein bound to a polynucleotide is likely to be more     stable. -   vi. The excess loading moieties can act as a control for the system     being set up correctly (i.e. is the ATP in the buffer etc). -   vii. The user can control the order of the one or more     polynucleotide binding proteins so that they are loaded in the     correct sequence on one loading moiety. -   viii. The user can control which polynucleotide binding proteins are     attached to which loading moiety, e.g. Y adaptor versus bridging     moiety. -   ix. The one or more polynucleotide binding proteins can be used to     purify the one or more loading moieties. -   x. The user can make different modifications to the polynucleotide     binding proteins attached to different loading moieties. -   xi. Different polynucleotide binding proteins may prefer different     binding conditions and so it may helpful to be able to pre-load them     on different loading moieties (rather than together on the target     polynucleotide). -   xii If the one or more polynucleotide binding proteins are     pre-loaded, there should not be any free polynucleotide binding     proteins after attachment and so the proteins can be used to purify     the construct. -   xiii The invention minimises use of the one or more polynucleotide     binding proteins and so there is less wastage. -   xiv The user can control where the one or more polynucleotide     binding proteins bind or do not bind with respect to the target     polynucleotide. By pre-loading the one or more polynucleotide     binding proteins on the one or more loading moieties, the one or     more polynucleotide binding proteins do not bind directly to the     target polynucleotide. -   xv The invention also improves the yield of sequence information,     eg. when a polymerase copys the target strand. Yields can be limited     by inefficient protein binding, and also lack of processivity.     Preloading overcomes loading inefficiencies, and creating a     closed-complex overcomes processivity problems.

The invention also provides a method of characterising a target polynucleotide. Once the one or more polynucleotide binding proteins have been loaded onto the target polynucleotide, they can be contacted with a transmembrane pore such that the one or more polynucleotide binding proteins control the movement of the polynucleotide with respect to the pore, such as through the pore. The method also comprises taking one or more measurements as the polynucleotide moves with respect to the pore. The measurements are indicative of one or more characteristics of the polynucleotide, such as the sequence.

It has been shown that double stranded polynucleotides can be effectively characterised using a transmembrane pore if they are modified to include a Y adaptor (a double stranded stem and two non-complementary arms) containing a leader sequence and a bridging moiety adaptor, such as a hairpin loop adaptor (WO 2013/014451). It is preferred that that Y adaptor containing the leader sequence is attached to one end of the polynucleotide and the bridging moiety adaptor is attached to the other end. The leader sequence preferentially threads into the nanopore and the bridging moiety (such as a hairpin loop) connecting the two strands of the polynucleotide allows both strands to be investigated as the polynucleotide unzips and both strands (connected via the bridging moiety) move with respect to the pore, such as through the pore. This is advantageous because it doubles the amount of information obtained from a single double-stranded polynucleotide. Moreover, because the sequences in the two strands are complementary, the information from the two strands can be combined informatically. This mechanism provides an orthogonal proof-reading capability that provides higher confidence observations. The one or more loading moieties used in accordance with the invention may be the Y adaptor and/or the bridging moiety adaptor. This is discussed in more detail below.

The invention also provides method of preparing a target polynucleotide for characterisation. The method may be for improving the target polynucleotide for characterisation. The method may be for modifying or extending the target polynucleotide. Once one or more polymerases have been loaded onto the target polynucleotide, they can be allowed to form one or more polynucleotides using the target polynucleotide as a template. If the target polynucleotide is single stranded, another complementary polynucleotide is formed. If the target polynucleotide is double stranded, both strands are preferably used as templates by the one or more polymerases. Because the strand(s) from the target polynucleotide and the new polynucleotide(s) produced by the one or more polymerases are complementary, the information from them can be combined informatically as discussed above. This type of method is also disclosed in WO 2013/014451. The polynucleotide(s) formed by the polymerase may comprise the same type of polynucleotide as the target polynucleotide or a different type of polynucleotide as discussed in more detail below. The target polynucleotide may be modified as discussed in more detail below. The one or more polymerases may be loaded onto the target polynucleotide using a Y adaptor and/or a bridging moiety adaptor as discussed in more detail below.

Polynucleotide

The target polynucleotide may be any polynucleotide. A polynucleotide, such as a nucleic acid, is a macromolecule comprising two or more nucleotides. The polynucleotide or nucleic acid may comprise any combination of any nucleotides. The nucleotides can be naturally occurring or artificial. One or more nucleotides in the polynucleotide can be oxidized or methylated. One or more nucleotides in the polynucleotide may be damaged. For instance, the polynucleotide may comprise a pyrimidine dimer. Such dimers are typically associated with damage by ultraviolet light and are the primary cause of skin melanomas. One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag. Suitable labels are described below. The polynucleotide may comprise one or more spacers.

A nucleotide typically contains a nucleobase, a sugar and at least one phosphate group. The nucleobase and sugar form a nucleoside. The nucleotide may be a natural nucleotide or a non-natural nucleotide.

The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C).

The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably a deoxyribose.

The nucleotide in the polynucleotide is typically a ribonucleotide or deoxyribonucleotide. The polynucleotide may comprise the following nucleosides: adenosine, uridine, guanosine and cytidine. The nucleotide is preferably a deoxyribonucleotide. The polynucleotide preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and/or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC).

The nucleotide typically contains a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5′ or 3′ side of a nucleotide.

Suitable nucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP) and deoxycytidine monophosphate (dCMP). The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP and dUMP. The nucleotides are most preferably selected from dAMP, dTMP, dGMP, dCMP and dUMP. The polynucleotide preferably comprises the following nucleotides: dAMP, dUMP and/or dTMP, dGMP and dCMP.

The nucleotides in the polynucleotide may be attached to each other in any manner. The nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids. The nucleotides may be connected via their nucleobases as in pyrimidine dimers.

The polynucleotide may be single stranded or double stranded. At least a portion of the polynucleotide is preferably double stranded.

The polynucleotide can be a nucleic acid. The polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), bridged nucleic acid (BNA) or other synthetic polymers with nucleotide side chains. The PNA backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA backbone is composed of repeating glycol units linked by phosphodiester bonds. The TNA backbone is composed of repeating threose sugars linked together by phosphodiester bonds. LNA is formed from ribonucleotides as discussed above having an extra bridge connecting the 2′ oxygen and 4′ carbon in the ribose moiety. Bridged nucleic acids (BNAs) are modified RNA nucleotides. They may also be called constrained or inaccessible RNA. BNA monomers can contain a five-membered, six-membered or even a seven-membered bridged structure with a “fixed” C3′-endo sugar puckering. The bridge is synthetically incorporated at the 2′, 4′-position of the ribose to produce a 2′, 4′-BNA monomer.

The polynucleotide is most preferably ribonucleic nucleic acid (RNA) or deoxyribonucleic acid (DNA).

The polynucleotide may be any length. For example, the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400 or at least 500 nucleotides in length. The polynucleotide can be 1000 or more nucleotides, 5000 or more nucleotides in length or 100000 or more nucleotides in length.

The helicase may move along the whole or only part of the polynucleotide in the method of the invention. The whole or only part of the target polynucleotide may be characterised using the method of the invention.

The polynucleotide may be single stranded. At least a portion of the polynucleotide is preferably double stranded. Helicases typically bind to single stranded polynucleotides. If at least a portion of the polynucleotide is double stranded, the polynucleotide preferably comprises a single stranded region or a non-hybridised region. The one or more helicases are capable of binding to the single stranded region or one strand of the non-hybridised region. The polynucleotide preferably comprises one or more single stranded regions or one or more non-hybridised regions.

The one or more spacers are preferably included in the single stranded region or the non-hybridised region of the polynucleotide. The polynucleotide may comprise more than one single stranded region or more than one non-hybridised region. The polynucleotide may comprise a single stranded region or a non-hybridised region within its sequence and/or at one or both ends. The one or more spacers may be included in the double stranded region of the polynucleotide.

If the one or more helicases used in the method move in the 5′ to 3′ direction, the polynucleotide preferably comprises a single stranded region or a non-hybridised region at its 5′ end. If the one or more helicases used in the method move in the 3′ to 5′ direction, the polynucleotide preferably comprises a single stranded region or a non-hybridised region at its 3′ end. If the one or more helicases are used in the inactive mode (i.e. as a brake), it does not matter where the single stranded region or the non-hybridised region is located.

The single stranded region preferably comprises a leader sequence which preferentially threads into the pore. This is discussed in more detail below.

If at least a portion of the polynucleotide is double stranded, the two strands of the double stranded portion are preferably linked using a bridging moiety, such as a hairpin or a hairpin loop. This facilitates characterisation method of the invention and is discussed in more detail below.

The polynucleotide is present in any suitable sample. The invention is typically carried out on a sample that is known to contain or suspected to contain the polynucleotide. The invention may be carried out on a sample to confirm the identity of one or more polynucleotides whose presence in the sample is known or expected.

The sample may be a biological sample. The invention may be carried out in vitro on a sample obtained from or extracted from any organism or microorganism. The organism or microorganism is typically archaeal, prokaryotic or eukaryotic and typically belongs to one of the five kingdoms: plantae, animalia, fungi, monera and protista. The invention may be carried out in vitro on a sample obtained from or extracted from any virus. The sample is preferably a fluid sample. The sample typically comprises a body fluid of the patient. The sample may be urine, lymph, saliva, mucus or amniotic fluid but is preferably blood, plasma or serum. Typically, the sample is human in origin, but alternatively it may be from another mammal animal such as from commercially farmed animals such as horses, cattle, sheep, fish, chickens or pigs or may alternatively be pets such as cats or dogs. Alternatively, the sample may be of plant origin, such as a sample obtained from a commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats, canola, maize, soya, rice, rhubarb, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa, cotton.

The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample. Examples of a non-biological sample include surgical fluids, water such as drinking water, sea water or river water, and reagents for laboratory tests.

The sample is typically processed prior to being used in the invention, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample may be measured immediately upon being taken. The sample may also be typically stored prior to assay, preferably below −70° C.

Polynucleotide Binding Proteins

A polynucleotide binding protein may be any protein that is capable of binding to the polynucleotide and controlling its movement with respect to the pore, such as through the pore. It is straightforward in the art to determine whether or not a protein binds to a polynucleotide. The protein typically interacts with and modifies at least one property of the polynucleotide. The protein may modify the polynucleotide by cleaving it to form individual nucleotides or shorter chains of nucleotides, such as di- or trinucleotides. The moiety may modify the polynucleotide by orienting it or moving it to a specific position, i.e. controlling its movement.

Any number of polynucleotide proteins may be attached to the target polynucleotide. For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more proteins may be attached.

The one or more polynucleotide binding proteins may be one or more single stranded binding proteins (SSBs). The one or more single-stranded binding proteins (SSBs) may comprise a carboxy-terminal (C-terminal) region which does not have a net negative charge or (ii) a modified SSB comprising one or more modifications in its C-terminal region which decreases the net negative charge of the C-terminal region. The one or more polynucleotide binding proteins may be any of the SSBs disclosed in International Application No. PCT/GB2013/051924 (published as WO 2014/013259).

The one or more polynucleotide binding proteins are preferably derived from a polynucleotide handling enzyme. A polynucleotide handling enzyme is a polypeptide that is capable of interacting with and modifying at least one property of a polynucleotide. The enzyme may modify the polynucleotide by cleaving it to form individual nucleotides or shorter chains of nucleotides, such as di- or trinucleotides. The enzyme may modify the polynucleotide by orienting it or moving it to a specific position. The polynucleotide handling enzyme does not need to display enzymatic activity as long as it is capable of binding the polynucleotide and controlling its movement with respect to the pore, such as through the pore. For instance, the enzyme may be modified to remove its enzymatic activity or may be used under conditions which prevent it from acting as an enzyme. Such conditions are discussed in more detail below.

The one or more polynucleotide binding proteins are preferably derived from a nucleolytic enzyme. The enzyme is more preferably derived from a member of any of the Enzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. The enzyme may be any of those disclosed in International Application No. PCT/GB10/000133 (published as WO 2010/086603).

Preferred enzymes are polymerases, exonucleases, helicases and topoisomerases, such as gyrases, and reverse transcriptases. Suitable enzymes include, but are not limited to, exonuclease I from E. coli (SEQ ID NO: 11), exonuclease III enzyme from E. coli (SEQ ID NO: 13), RecJ from T. thermophilus (SEQ ID NO: 15) and bacteriophage lambda exonuclease (SEQ ID NO: 17), TatD exonuclease and variants thereof. Three subunits comprising the sequence shown in SEQ ID NO: 15 or a variant thereof interact to form a trimer exonuclease. The polymerase may be PyroPhage® 3173 DNA Polymerase (which is commercially available from Lucigen® Corporation), SD Polymerase (commercially available from Bioron®), Klenow from NEB or variants thereof. The enzyme is preferably Phi29 DNA polymerase (SEQ ID NO: 9) or a variant thereof. Preferred versions of Phi29 are discussed in more detail below. Modified versions of Phi29 polymerase that may be used in the invention are discussed below and disclosed in U.S. Pat. No. 5,576,204. The topoisomerase is preferably a member of any of the Moiety Classification (EC) groups 5.99.1.2 and 5.99.1.3. Reverse transcriptases are enzymes which are capable of catalysing the formation of cDNA from a RNA template. They are commercially available from, for instance, New England Biolabs® and Invitrogen®.

The one or more polynucleotide binding proteins are preferably derived from a helicase. Helicases can control the movement of polynucleotides in at least two active modes of operation (when the helicase is provided with all the necessary components to facilitate movement, e.g. ATP and Mg²⁺) and one inactive mode of operation (when the helicase is not provided with the necessary components to facilitate movement or is modified to prevent or hinder movement). When provided with all the necessary components to facilitate movement, the helicase moves along the polynucleotide in a 5′ to 3′ or a 3′ to 5′ direction (depending on the helicase), but the orientation of the polynucleotide in the pore (which is dependent on which end of the polynucleotide is captured by the pore) means that the helicase can be used to either move the polynucleotide out of the pore against the applied field or move the polynucleotide into the pore with the applied field. When the end of the polynucleotide towards which the helicase moves is captured by the pore, the helicase works against the direction of the field resulting from the applied potential and pulls the threaded polynucleotide out of the pore and into the cis chamber. However, when the end away from which the helicase moves is captured in the pore, the helicase works with the direction of the field resulting from the applied potential and pushes the threaded polynucleotide into the pore and into the trans chamber.

When the helicase is not provided with the necessary components to facilitate movement it can bind to the polynucleotide and act as a brake slowing the movement of the polynucleotide when it is pulled into the pore by the field resulting from the applied potential. In the inactive mode, it does not matter which end of the polynucleotide is captured, it is the applied field which pulls the polynucleotide into the pore towards the trans side with the helicase acting as a brake. When in the inactive mode, the movement control of the polynucleotide by the helicase can be described in a number of ways including ratcheting, sliding and braking.

In the characterization method of the invention, the one or more helicases preferably control the movement of the target polynucleotide with respect to the pore, such as through the pore, with the field resulting from the applied potential. In one preferred embodiment, the one or more helicases are used in the active mode and the end away from which the one or more helicases move is captured by the pore such that the one or more helicases work with the field resulting from the applied potential and push the polynucleotide with respect to the pore, such as through the pore. If the one or more helicases move in the 5′ to 3′ direction, the 5′ end of the polynucleotide is preferably captured by the pore. In such embodiments, the one or more helicases move along the polynucleotide in the 5′ to 3′ direction. If the one or more helicases move in the 3′ to 5′ direction, the 3′ end of the polynucleotide is preferably captured by the pore. In such embodiments, the one or more helicases move along the polynucleotide in the 3′ to 5′ direction.

In another preferred embodiment of the characterization method, the one or more helicases are used in the inactive mode such that the applied field pulls the polynucleotide with respect to the pore, such as through the pore, and the one or more helicases act as a brake. In another preferred embodiment, the one or more helicases are modified such that they retain their polynucleotide binding ability but lack helicase activity (i.e. the ability to actively move along the polynucleotide) such that the applied field pulls the polynucleotide with respect to the pore, such as through the pore, and the one or more helicases act as a brake. In the method of the invention, the one or more helicases preferably slow or brake the movement of the polynucleotide with respect to the pore, such as through the pore, with the field resulting from the applied potential. In either case, the one or more helicases are typically too large to move with respect to the pore, such as through the pore, and the pore pushes the one or more helicases along the polynucleotide as the polynucleotide moves with respect to the pore, such as through the pore, with the field resulting from the applied potential.

Any steps in the characterization method using one or more helicases are typically carried out in the presence of free nucleotides or free nucleotide analogues and an enzyme cofactor that facilitates the action of the one or more helicases. The free nucleotides may be one or more of any of the individual nucleotides discussed above. The free nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP). The free nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotides are preferably adenosine triphosphate (ATP). The enzyme cofactor is a factor that allows the construct to function. The enzyme cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg²⁺, Mn²⁺, Ca²⁺ or Co²⁺. The enzyme cofactor is most preferably Mg²⁺.

Any helicase may be used in the invention. The helicase may be or be derived from a He1308 helicase, a RecD helicase, such as TraI helicase or a TrwC helicase, a XPD helicase or a Dda helicase. The helicase may be any of the helicases, modified helicases or helicase constructs disclosed in International Application Nos. PCT/GB2012/052579 (published as WO 2013/057495); PCT/GB2012/053274 (published as WO 2013/098562); PCT/GB2012/053273 (published as WO2013098561); PCT/GB2013/051925 (published as WO 2014/013260); PCT/GB2013/051924 (published as WO 2014/013259) and PCT/GB2013/051928 (published as WO 2014/013262); and in International Application No. PCT/GB2014/052736.

The one or more polynucleotide binding proteins may be derived from a helicase, such as He1308 Mbu (SEQ ID NO: 18), He1308 Csy (SEQ ID NO: 19), He1308 Tga (SEQ ID NO: 20), He1308 Mhu (SEQ ID NO: 21), TraI Eco (SEQ ID NO: 22), XPD Mbu (SEQ ID NO: 23) or a variant thereof. The one or more polynucleotide binding proteins preferably comprise the sequence shown in SEQ ID NO: 25 (Trwc Cba) or a variant thereof, the sequence shown in SEQ ID NO: 18 (He1308 Mbu) or a variant thereof or the sequence shown in SEQ ID NO: 24 (Dda) or a variant thereof. Variants may differ from the native sequences in any of the ways discussed below for helicases or transmembrane pores.

A preferred variant of SEQ ID NO: 24 comprises (or only comprises) (a) E94C/A360C, (b) E94C/A360C and then (ΔM1)G1G2 (i.e. deletion of M1 and then addition G1 and G2), (c) E94C/A360C/C109A/C136A or (d) E94C/A360C/C109A/C136A and then (ΔM1)G1G2 (i.e. deletion of M1 and then addition G1 and G2).

Other preferred variants of SEQ ID NO: 24 comprise W378A. Preferred variants of SEQ ID NO: 24 comprise (or comprise only) (a) E94C/A360C/W378A, (b) E94C/A360C/W378A and then (ΔM1)G1G2 (i.e. deletion of M1 and then addition G1 and G2), (c) E94C/A360C/C109A/C136A/W378A or (d) E94C/A360C/C109A/C136A/W378A and then (ΔM1)G1G2 (i.e. deletion of M1 and then addition G1 and G2).

Preferred variants of SEQ ID NO: 25 comprises (or only comprises) (a) Q594A, (b) L376C/Q594A/K762C, (c) L376C/Q594A/A779C, (d) Q346C/Q594A/A779C, (e) Q346C/Q594A/A783C, (f) D411/Q594A/A783C, (g) Q594A/R353C/E722C, (h) Q594A/Q357C/T720C, (i) Q594A/R358C/T720C, (j) Q594A/H354C/T720C, (k) Q594A/F374C/E722C or (1) Q594A/S350C/E722C. Any of (a) to (1) may further comprise and then (ΔM1)G1G2 (i.e. deletion of M1 and then addition G1 and G2). Other Preferred variants are discussed above.

The one or more helicases are preferably modified to reduce the size of an opening in the polynucleotide binding domain through which in at least one conformational state the polynucleotide can unbind from the helicase. The one or more helicases are preferably modified to close an opening in the polynucleotide binding domain through which in at least one conformational state the polynucleotide can unbind from the helicase. This is disclosed in WO 2014/013260 and PCT/GB2014/052736. Any of the modifications disclosed in WO 2014/013260 and PCT/GB2014/052736 may be used in the invention.

The ability of a helicase to bind to and unbind from a polynucleotide can be determined using any method known in the art. Suitable binding/unbinding assays include, but are not limited to, native polyacrylamide gel electrophoresis (PAGE), fluorescence anisotropy, calorimetry and Surface plasmon resonance (SPR, such as Biacore™). The ability of a helicase to unbind from a polynucleotide can of course be determined by measuring the time for which the helicase can control the movement of a polynucleotide. This may also be determined using any method known in the art. The ability of a helicase to control the movement of a polynucleotide is typically assayed in a nanopore system, such as the ones described below. The ability of a helicase to control the movement of a polynucleotide can be determined as described in the Examples.

As disclosed in PCT/GB2014/052736, a helicase used in the invention may be a Dda helicase in which at least one cysteine residue and/or at least one non-natural amino acid have been introduced into the hook domain and/or the 2A (RecA-like motor) domain, wherein the helicase retains its ability to control the movement of a polynucleotide. Any number of cysteine residues and/or non-natural amino acids may be introduced into each domain. For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cysteine residues may be introduced and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-natural amino acids may be introduced. Only one or more cysteine residues may be introduced. Only one or more non-natural amino acids may be introduced. A combination of one or more cysteine residues and one or more non-natural amino acids may be introduced. The at least one cysteine residue and/or at least one non-natural amino acid are/is preferably introduced by substitution. Methods for doing this are known in the art. These Dda modifications do not prevent the helicase from binding to a polynucleotide. These modifications decrease the ability of the polynucleotide to unbind or disengage from the helicase. In other words, the one or more modifications increase the processivity of the Dda helicase by preventing dissociation from the polynucleotide strand. The thermal stability of the enzyme is typically also increased by the one or more modifications giving it an improved structural stability that is beneficial in Strand Sequencing. A non-natural amino acid is an amino that is not naturally found in a Dda helicase. The non-natural amino acid is preferably not histidine, alanine, isoleucine, arginine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine or tyrosine. The non-natural amino acid is more preferably not any of the twenty amino acids in the previous sentence or selenocysteine

Preferred non-natural amino acids for use in the invention include, but are not limited, to 4-Azido-L-phenylalanine (Faz), 4-Acetyl-L-phenylalanine, 3-Acetyl-L-phenylalanine, 4-Acetoacetyl-L-phenylalanine, O-Allyl-L-tyrosine, 3-(Phenylselanyl)-L-alanine, O-2-Propyn yl-L-tyrosine, 4-(Dihydroxyboryl)-L-phenylalanine, 4-[(Ethylsulfanyl)carbonyl]-L-phenylalanine, (2S)-2-amino-3-{4-[(propan-2-ylsulfanyl)carbonyl]phenyl}propanoic acid, (2S)-2-amino-3-{4-[(2-amino-3-sulfanylpropanoyl)amino]phenyl}propanoic acid, O-Methyl-L-tyrosine, 4-Amino-L-phenylalanine, 4-Cyano-L-phenylalanine, 3-Cyano-L-phenylalanine, 4-Fluoro-L-phenylalanine, 4-Iodo-L-phenylalanine, 4-Bromo-L-phenylalanine, O-(Trifluoromethyl)tyrosine, 4-Nitro-L-phenylalanine, 3-Hydroxy-L-tyrosine, 3-Amino-L-tyrosine, 3-Iodo-L-tyrosine, 4-Isopropyl-L-phenylalanine, 3-(2-Naphthyl)-L-alanine, 4-Phenyl-L-phenylalanine, (2S)-2-amino-3-(naphthalen-2-ylamino)propanoic acid, 6-(Methylsulfanyl)norleucine, 6-Oxo-L-lysine, D-tyrosine, (2R)-2-Hydroxy-3-(4-hydroxyphenyl)propanoic acid, (2R)-2-Ammoniooctanoate3-(2,2′-Bipyridin-5-yl)-D-alanine, 2-amino-3-(8-hydroxy-3-quinolyl)propanoic acid, 4-Benzoyl-L-phenylalanine, S-(2-Nitrobenzyl)cysteine, (2R)-2-amino-3-[(2-nitrobenzyl)sulfanyl]propanoic acid, (2S)-2-amino-3-[(2-nitrobenzyl)oxy]propanoic acid, O-(4,5-Dimethoxy-2-nitrobenzyl)-L-serine, (2S)-2-amino-6-({[(2-nitrobenzyl)oxy]carbonyl}amino)hexanoic acid, O-(2-Nitrobenzyl)-L-tyrosine, 2-Nitrophenylalanine, 4-[(E)-Phenyldiazenyl]-L-phenylalanine, 4-[3-(Trifluoromethyl)-3H-diaziren-3-yl]-D-phenylalanine, 2-amino-3-[[5-(dimethylamino)-1-naphthyl]sulfonylamino]propanoic acid, (2S)-2-amino-4-(7-hydroxy-2-oxo-2H-chromen-4-yl)butanoic acid, (2S)-3-[(6-acetylnaphthalen-2-yl)amino]-2-aminopropanoic acid, 4-(Carboxymethyl)phenylalanine, 3-Nitro-L-tyrosine, O-Sulfo-L-tyrosine, (2R)-6-Acetamido-2-ammoniohexanoate, 1-Methylhistidine, 2-Aminononanoic acid, 2-Aminodecanoic acid, L-Homocysteine, 5-Sulfanylnorvaline, 6-Sulfanyl-L-norleucine, 5-(Methylsulfanyl)-L-norvaline, N⁶-{[(2R,3R)-3-Methyl-3,4-dihydro-2H-pyrrol-2-yl]carbonyl}-L-lysine, N⁶-[(Benzyloxy)carbonyl]lysine, (2S)-2-amino-6-[(cyclopentylcarbonyl)amino]hexanoic acid, N⁶-[(Cyclopentyloxy)carbonyl]-L-lysine, (2S)-2-amino-6-{[(2R)-tetrahydrofuran-2-ylcarbonyl]amino}hexanoic acid, (2S)-2-amino-8-[(2R,3S)-3-ethynyltetrahydrofuran-2-yl]-8-oxooctanoic acid, N⁶-(tert-Butoxycarbonyl)-L-lysine, (2S)-2-Hydroxy-6-({[(2-methyl-2-propanyl)oxy]carbonyl}amino)hexanoic acid, N⁶-[(Allyloxy)carbonyl]lysine, (2S)-2-amino-6-({[(2-azidobenzyl)oxy]carbonyl}amino)hexanoic acid, N⁶-L-Prolyl-L-lysine, (2S)-2-amino-6-{[(prop-2-yn-1-yloxy)carbonyl]amino}hexanoic acid and N⁶-[(2-Azidoethoxy)carbonyl]-L-lysine. The most preferred non-natural amino acid is 4-azido-L-phenylalanine (Faz).

The helicase used in the invention preferably comprises a variant of SEQ ID NO: 24 in which at least one cysteine residue and/or at least one non-natural amino acid have been introduced into (i) the tower domain (residues D260-P274 and N292-A389) and/or (ii) the pin domain (residues K86-E102) and/or the (iii) 1A domain (residues M1-L85 and V103-K177). The at least one cysteine residue and/or at least one non-natural amino acid are preferably introduced into residues N292-A389 of the tower domain.

The introductions of at least two cysteines into SEQ ID NOs: 24 and 25 as discussed above reduces the size of or closes an opening in the polynucleotide binding domain of the helicases.

Preferred helicase constructs for use in the invention are described in International Application Nos. PCT/GB2013/051925 (published as WO 2014/013260); PCT/GB2013/051924 (published as WO 2014/013259) and PCT/GB2013/051928 (published as WO 2014/013262); and in UK Application No. 1318464.3 filed on 18 Oct. 2013.

A variant of SEQ ID NOs: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25 is an enzyme that has an amino acid sequence which varies from that of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25 and which retains polynucleotide binding ability. This can be measured using any method known in the art. For instance, the variant can be contacted with a polynucleotide and its ability to bind to and move along the polynucleotide can be measured. The variant may include modifications that facilitate binding of the polynucleotide and/or facilitate its activity at high salt concentrations and/or room temperature. Variants may be modified such that they bind polynucleotides (i.e. retain polynucleotide binding ability) but do not function as an enzyme. For instance, variants of helicases may be modified such that they bind polynucleotides (i.e. retain polynucleotide binding ability) but do not function as a helicase (i.e. do not move along polynucleotides when provided with all the necessary components to facilitate movement, e.g. ATP and Mg²⁺). Such modifications are known in the art. For instance, modification of the Mg²⁺ binding domain in helicases typically results in variants which do not function as helicases. These types of variants may act as molecular brakes. A preferred molecular brake is TrwC Cba-Q594A (SEQ ID NO: 25 with the mutation Q594A). Others are discussed above with reference to SEQ ID NO: 25. This variant does not function as a helicase (i.e. binds to polynucleotides but does not move along them when provided with all the necessary components to facilitate movement, e.g. ATP and Mg²⁺). The one or more molecular brake helicases can be used in any direction and/or mode discussed above.

Over the entire length of the amino acid sequence of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25, a variant will preferably be at least 50% homologous to that sequence based on amino acid identity. More preferably, the variant polypeptide may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 9, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a stretch of 200 or more, for example 230, 250, 270, 280, 300, 400, 500, 600, 700, 800, 900 or 1000 or more, contiguous amino acids (“hard homology”). Homology is determined as described above. The variant may differ from the wild-type sequence in any of the ways discussed above with reference to SEQ ID NO: 2 and 4 below.

If two or more polynucleotide binding proteins are used, they may be the same or different. Any combination of the proteins discussed above may be used. For instance, the two or more proteins may be different variants of the same protein, such as helicase. The two or more polynucleotide binding proteins preferably comprise one or more helicases and one or more polymerases.

If two or more polynucleotide binding proteins are used, they may be attached to one another. The two or more polynucleotide binding proteins may be covalently attached to one another. The polynucleotide binding proteins may be attached in any order and using any method. Preferred helicase constructs for use in the invention are described in International Application Nos. PCT/GB2013/051925 (published as WO 2014/013260); PCT/GB2013/051924 (published as WO 2014/013259) and PCT/GB2013/051928 (published as WO 2014/013262); and in UK Application No. 1318464.3 filed on 18 Oct. 2013.

If two or more polynucleotide binding proteins are used, they are preferably not attached to one another except via the polynucleotide. The two or more polynucleotide binding proteins are more preferably not covalently attached to one another.

One or More Helicases and One or More Molecular Brakes

In some instances the characterisation method of the invention may concern using one or more helicases and one or more molecular brakes. When the target polynucleotide is contacted with the pore, the one or more helicases and the one or more molecular brakes are brought together and both control the movement of the polynucleotide with respect to the pore, such as through the pore.

The one or more helicases may be any of those discussed above. The one or more molecular brakes may be any compound or molecule which binds to the polynucleotide and slows the movement of the polynucleotide with respect to the pore, such as through the pore. The one or more molecular brakes preferably comprise one or more compounds which bind to the polynucleotide. The one or more compounds are preferably one or more macrocycles. Suitable macrocycles include, but are not limited to, cyclodextrins, calixarenes, cyclic peptides, crown ethers, cucurbiturils, pillararenes, derivatives thereof or a combination thereof. The cyclodextrin or derivative thereof may be any of those disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J. Am. Chem. Soc. 116, 6081-6088. The agent is more preferably heptakis-6-amino-β-cyclodextrin (am₇-βCD), 6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) or heptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD).

The one or more molecular brakes are preferably one or more single stranded binding proteins (SSB). The one or more molecular brakes are more preferably a single-stranded binding protein (SSB) comprising a carboxy-terminal (C-terminal) region which does not have a net negative charge or (ii) a modified SSB comprising one or more modifications in its C-terminal region which decreases the net negative charge of the C-terminal region. The one or more molecular brakes are most preferably any of the SSBs disclosed in International Application No. PCT/GB2013/051924 (published as WO 2014/013259).

The one or more molecular brakes are preferably one or more polynucleotide binding proteins. The polynucleotide binding protein may be any protein that is capable of binding to the polynucleotide and controlling its movement with respect to the pore, such as through the pore. It is straightforward in the art to determine whether or not a protein binds to a polynucleotide. The protein typically interacts with and modifies at least one property of the polynucleotide. The protein may modify the polynucleotide by cleaving it to form individual nucleotides or shorter chains of nucleotides, such as di- or trinucleotides. The moiety may modify the polynucleotide by orienting it or moving it to a specific position, i.e. controlling its movement.

The polynucleotide binding protein is preferably derived from a polynucleotide handling enzyme. The one or more molecular brakes may be derived from any of the polynucleotide handling enzymes discussed above. Modified versions of Phi29 polymerase (SEQ ID NO: 9) which act as molecular brakes are disclosed in U.S. Pat. No. 5,576,204. Modified versions of Phi29 polymerase (SEQ ID NO: 9) which act as molecular brakes are disclosed below. The one or more molecular brakes are preferably derived from a helicase.

Any number of molecular brakes derived from a helicase may be used. For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases may be used as molecular brakes. If two or more helicases are be used as molecular brakes, the two or more helicases are typically the same helicase. The two or more helicases may be different helicases.

The two or more helicases may be any combination of the helicases mentioned above. The two or more helicases may be two or more Dda helicases. The two or more helicases may be one or more Dda helicases and one or more TrwC helicases. The two or more helicases may be different variants of the same helicase.

The two or more helicases are preferably attached to one another. The two or more helicases are more preferably covalently attached to one another. The helicases may be attached in any order and using any method. The one or more molecular brakes derived from helicases are preferably modified to reduce the size of an opening in the polynucleotide binding domain through which in at least one conformational state the polynucleotide can unbind from the helicase. This is disclosed in WO 2014/013260.

Preferred helicase constructs for use in the invention are described in International Application Nos. PCT/GB2013/051925 (published as WO 2014/013260); PCT/GB2013/051924 (published as WO 2014/013259); PCT/GB2013/051928 (published as WO 2014/013262) and PCT/GB2014/052736.

If the one or more helicases are used in the active mode (i.e. when the one or more helicases are provided with all the necessary components to facilitate movement, e.g. ATP and Mg²⁺), the one or more molecular brakes are preferably (a) used in an inactive mode (i.e. are used in the absence of the necessary components to facilitate movement or are incapable of active movement), (b) used in an active mode where the one or more molecular brakes move in the opposite direction to the one or more helicases or (c) used in an active mode where the one or more molecular brakes move in the same direction as the one or more helicases and more slowly than the one or more helicases.

If the one or more helicases are used in the inactive mode (i.e. when the one or more helicases are not provided with all the necessary components to facilitate movement, e.g. ATP and Mg²⁺ or are incapable of active movement), the one or more molecular brakes are preferably (a) used in an inactive mode (i.e. are used in the absence of the necessary components to facilitate movement or are incapable of active movement) or (b) used in an active mode where the one or more molecular brakes move along the polynucleotide in the same direction as the polynucleotide with respect to the pore, such as through the pore.

The one or more helicases and one or more molecular brakes may be attached to the polynucleotide at any positions so that they are brought together and both control the movement of the polynucleotide with respect to the pore, such as through the pore. The one or more helicases and one or more molecular brakes are at least one nucleotide apart, such as at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 50,000 nucleotides or more apart. If the method concerns characterising a double stranded polynucleotide provided with a Y adaptor at one end and a bridging moiety adaptor, such as a hairpin loop adaptor, at the other end, the one or more helicases are preferably attached to the Y adaptor and the one or more molecular brakes are preferably attached to the bridging moiety adaptor. In this embodiment, the one or more molecular brakes are preferably one or more helicases that are modified such that they bind the polynucleotide but do not function as a helicase. The one or more helicases attached to the Y adaptor are preferably stalled at a spacer as discussed in more detail below. The one or more molecular brakes attached to the bridging moiety adaptor are preferably not stalled at a spacer. The one or more helicases and the one or more molecular brakes are preferably brought together when the one or more helicases reach the bridging moiety. The one or more helicases may be attached to the Y adaptor before the Y adaptor is attached to the polynucleotide or after the Y adaptor is attached to the polynucleotide. The one or more molecular brakes may be attached to the bridging moiety adaptor before the bridging moiety adaptor is attached to the polynucleotide or after the bridging moiety adaptor is attached to the polynucleotide.

The one or more helicases and the one or more molecular brakes are preferably not attached to one another. The one or more helicases and the one or more molecular brakes are more preferably not covalently attached to one another. The one or more helicases and the one or more molecular brakes are preferably not attached as described in International Application Nos. PCT/GB2013/051925 (published as WO 2014/013260); PCT/GB2013/051924 (published as WO 2014/013259) and PCT/GB2013/051928 (published as WO 2014/013262); and in UK Application No. 1318464.3 filed on 18 Oct. 2013.

One or More Polymerases

The method of the invention preferably concerns attaching one or more polymerases to the target polynucleotide. Any of the polymerases discussed above or below may be used. For instance, the polymerase may comprise the sequence shown in SEQ ID NO: 9 or 31 or a variant thereof as defined above.

Preferred variants of SEQ ID NO: 9 include, but are not limited to, variants which comprise the following substitutions (a) G410C and P562C, (b) A411C and Q560C, (c) K402C and I94C, (d) A406C and E75C and (e) L412C and Q560C. The introduction of these cysteines into SEQ ID NO: 9 reduces the size of or closes an opening in the polynucleotide binding domain of the polymerase as discussed above for helicases. The variants in (a) to (e) may be used as molecular brakes as discussed above.

Once the one or more polymerases are loaded on the target polynucleotide, it/they may be allowed to form one or more polynucleotides using the target polynucleotide as a template. This typically involves contacting the target polynucleotide and the one or more polymerases with a population of free nucleotides under conditions in which the polymerase forms one or more polynucleotides using the target polynucleotide as a template. Suitable conditions are discussed below. Any number of polynucleotides may be formed by the one or more polymerases. One or two polynucleotides are preferably formed.

The population of free nucleotides may comprise any of the nucleotides discussed above and below. The nature of the one or more polynucleotides formed by the one or more polymerase will depend on the free nucleotides in the population. The one or more polymerases may form one or more polynucleotides of the same type as the target polynucleotide. For instance, if the target polynucleotide is DNA, the invention may use a population of free DNA nucleotides (i.e. nucleotides comprising deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine and deoxymethylcytidine) such that the one or more polymerases form one or more DNA polynucleotides using the target polynucleotide as a template. The one or more polymerases may form one or more polynucleotides of a different type from the target polynucleotide. For instance, if the target polynucleotide is RNA, the invention may use a population of free DNA nucleotides such that the one or more polymerases form one or more DNA polynucleotides using the target polynucleotide as a template. The one or more polymerases may modify the target polynucleotide as discussed in more detail below.

A primer or a 3′ hairpin is typically used as the nucleation point for polymerase extension. The one or more polymerases may be attached to the target polynucleotide in any of the ways discussed herein. The one or more polymerases are typically provided bound to one or more loading moieties, such as one or more Y adaptors and/or one or more bridging moiety adaptors (such as one or more hairpin loop adaptors).

The method of the invention preferably comprises attaching one or more polymerases to a single stranded target polynucleotide in accordance with the invention and allowing the one or more polymerases to form a polynucleotide using the target polynucleotide as a template and thereby producing a double stranded polynucleotide. The double stranded polynucleotide comprises the target polynucleotide (template) and complementary polynucleotide formed by the one or more polymerases. The two strands of the double stranded polynucleotide formed in this way may be linked with a bridging moiety adaptor, such as a hairpin loop adaptor, and both strands of the double stranded polynucleotide may be characterised as discussed below.

The invention provides a method of preparing the target polynucleotide for characterisation. The method comprises attaching one or more polymerases to the target polynucleotide using the invention and then using the one or more polymerases to produce one or more additional polynucleotides using the target polynucleotide as a template. The production of polynucleotides which complement the target polynucleotide facilitates its characterisation as discussed above.

The method preferably comprises attaching one or more polymerases to a double stranded target polynucleotide in accordance with the invention and allowing the one or more polymerases to form one or more polynucleotides using each strand of the target polynucleotide as a template.

In a preferred embodiment, the two strands of the double stranded target polynucleotide are linked at one end by a bridiging moiety adaptor (such as a hairpin loop adaptor) and the one or more polymerases are attached to the double stranded target polynucleotide at the other end using a loading moiety comprising another bridging moiety. The loading moiety may be a Y adaptor. The other bridging moiety is typically formed by a hairpin loop at the end of the strand of the Y adaptor to which the one or more polymerases are bound. The loading moiety may comprise one or more helicases, preferably bound to the opposite strand from the strand to which the one or more polymerases are bound. In this embodiment, the one or more polymerases will produce a double stranded construct in which the two strands of the construct are linked at one end by a bridging moiety and each strand of the construct comprises one strand of the target polynucleotide and a complementary polynucleotide formed by the one or more polymerases. This is shown in FIG. 17 . The two strands of the double stranded construct may be characterised as discussed below. In this embodiment, each strand of the original target polynucleotide is characterised twice. The one or polymerases may be used to control the movement of the double stranded construct with respect to the pore, such as through the pore. The one or more polymerases may be molecular brakes. If the loading moiety also comprises one or more helicases, the one or more helicases may be used to control the movement of the double stranded construct with respect to the pore, such as through the pore. In some embodiments, the one or more polymerases and the one or more helicases may both be used to control the movement of the double stranded construct with respect to the pore, such as through the pore.

In another embodiment, the two strands of the double stranded target polynucleotide are not linked at either end by a bridging moiety adaptor (such as a hairpin loop adaptor). One or more polymerases are attached to the double stranded target polynucleotide at each end using a loading moiety comprising a bridging moiety. The loading moiety at each end may be the same or different. Each loading moiety may be a Y adaptor. The bridiging moiety is typically formed by a hairpin loop at the end of the strand of the Y adaptor to which the one or more polymerases are bound. The loading moieties may comprise one or more helicases, preferably bound to the opposite strand from the strand to which the one or polymerases are bound. In this embodiment, the one or more polymerases will produce two double stranded constructs in which the two strands of each construct are linked at one end by a bridging moiety and each construct comprises one strand of the target polynucleotide and a complementary polynucleotide formed by the one or more polymerases. This is shown in FIG. 18 . The two constructs may be characterised as discussed below. In this embodiment, each strand of the original target polynucleotide is characterised twice, once in each construct. The one or polymerases may control the movement of each double stranded construct with respect to the pore, such as through the pore. The one or more polymerases may be molecular brakes. If each loading moiety also comprises one or more helicases, the one or more helicases may be used to control the movement of each double stranded construct with respect to the pore, such as through the pore. In some embodiments, the one or more polymerases and the one or more helicases may both be used to control the movement of each double stranded construct with respect to the pore, such as through the pore.

In another embodiment, the two strands of the double stranded target polynucleotide are linked at both ends by a bridiging moiety adaptor (such as a hairpin loop adaptor) to form a circular construct and one or more polymerases are attached to each bridging moiety adaptor. One or both of the bridging moiety adaptors may comprise one or more helicases, preferably bound to a polynucleotide which is hybridised to the bridging moiety adaptor. The double stranded polynucleotide formed by the hybridisation of this polynucleotide to the bridging adaptor moiety may form a nucleation for polymerase extension by the one or more polymerases. In this embodiment, the one or more polymerases at each end of the circular construct will use the circular construct as a template and produce a construct comprising multiple copies of each strand of the target polynucleotide. Two constructs will be produced from each target polynucleotide because there are two groups of one or more polymerases. This is shown in FIG. 19 . Each construct may be characterised as discussed below. Each construct will typically comprise multiple copies of each strand of the target polynucleotide. The copies in the construct may hybridise together to form double stranded regions. In this embodiment, each strand of the original target polynucleotide will be characterised as many times as the one or more polymerases copy them. The one or polymerases may control the movement of the construct with respect to the pore, such as through the pore. The one or more polymerases may be molecular brakes. If the bridging moiety adaptor also comprises one or more helicases, the one or more helicases may be used to control the movement of the construct with respect to the pore, such as through the pore. In some embodiments, the one or more polymerases and the one or helicases may both be used to control the movement of the construct with respect to the pore, such as through the pore.

After using the one or more polymerases to produce the one or more polylnucleotides, the method preferably comprises contacting the target polynucleotide and the one or more polynucleotides with a transmembrane pore such that the target polynucleotide and the one or more polynucleotides move with respect to the pore, such as through the pore. The method preferably comprises contacting the target polynucleotide and the one or more polynucleotides with a transmembrane pore such that the one or more polymerases control the movement of the target polynucleotide and the one or more polynucleotides with respect to the pore, such as through the pore. One or more helicases may also be used to control the movement of the target polynucleotide and the one or more polynucleotides with respect to the pore, such as through the pore. The one or more helicases may be attached to the target polynucleotide and the one or more polynucleotides as discussed above.

The method also comprises taking one or more measurements as the target polynucleotide and one or more polynucleotides move with respect to the pore wherein the measurements are indicative of one or more characteristics of the polynucleotides and thereby characterising the target polynucleotide. Any of the embodiments discussed below are applicable to this method.

Loading Moiety

The one or more polynucleotide binding proteins are provided bound to (or attached to) one or more loading moieties. In a preferred embodiment, the method further comprises binding (or attaching) the one or more polynucleotide binding proteins to the one or more loading moieties.

Each loading moiety may be any moiety that is capable of being attached to the target polynucleotide. Each loading moiety may be any length as long as the polynucleotide binding proteins may bind and it can be attached to the target polynucleotide.

The one or more loading moieties are preferably synthetic or artificial. The one or more loading moieties are preferably non-natural.

Suitable loading moieties include, but are not limited to a polymeric linker, a chemical linker, a polynucleotide or a polypeptide. The one or more loading moieties preferably comprise a polynucleotide or a loading polynucleotide. In such embodiments, the one or more polynucleotide binding proteins are preferably bound to (or attached to) the polynucleotide. Any of the polynucleotides discussed above may be used. Preferably, the one or more loading moieties comprise DNA, RNA, modified DNA (such as abasic DNA), RNA, PNA, LNA, BNA or PEG. The one or more loading moieties more preferably comprise single stranded or double stranded DNA or RNA.

The one or more loading moieties preferably comprise a single stranded polynucleotide to which the one or more polynucleotide binding proteins are bound (or attached).

At least one of the one or more loading moieties is preferably a Y adaptor. Y adaptors are defined in the section concerning double coupling and may comprise a leader sequence.

At least one of the one or more loading moieties is preferably a bridging moiety. The bridging moiety is most preferably a hairpin loop or a hairpin loop adaptor. Suitable hairpin loop adaptors can be designed using methods known in the art. The hairpin loop may be any length. If used as a loading moiety, the hairpin loop is typically 400 or fewer nucleotides, such as 350 or fewer nucleotides, 300 or fewer nucleotides, 250 or fewer nucleotides, 200 or fewer nucleotides, 150 or fewer nucleotides, 100 or fewer nucleotides, 90 or fewer nucleotides, 80 or fewer nucleotides, 70 or fewer nucleotides, 60 or fewer nucleotides, 50 or fewer nucleotides, 40 or fewer nucleotides, 30 or fewer nucleotides, 20 or fewer nucleotides or 10 or fewer nucleotides, in length. The hairpin loop is preferably from about 1 to 400, from 2 to 300, from 5 to 200, from 6 to 100 nucleotides in length. Hairpin loops are formed when two complementary parts of a polynucleotide hybridise to form a double stranded sequence (called a stem). If used as a loading moiety, the stem of the hairpin loop is preferably 200 or fewer nucleotide pairs, such as 150 or fewer nucleotide pairs, 100 or fewer nucleotide pairs, 90 or fewer nucleotide pairs, 80 or fewer nucleotide pairs, 70 or fewer nucleotide pairs, 60 or fewer nucleotide pairs, 50 or fewer nucleotide pairs, 40 or fewer nucleotide pairs, 30 or fewer nucleotide pairs, 20 or fewer nucleotide pairs or 10 or fewer nucleotide pairs, in length. The one or more polynucleotide binding proteins typically bind to the loop of the hairpin, i.e. not the stem.

If the target polynucleotide is double stranded, the one or more loading moieties preferably comprise a Y adaptor and optionally a bridging moiety, such as a hairpin loop adaptor. If at least one or more of the loading moieties is Y adaptor, it may be used in combination with a bridging adaptor that does not have any polynucleotide binding proteins bound or attached.

If the one or more polynucleotide binding proteins are derived from helicases, they may be stalled at one or more spacers on the one or more loading moieties. These are discussed in more detail below.

Any number of one or more loading moieties may be used. The method may comprise attaching two or more loading moieties each having one or more polynucleotide binding proteins bound (attached) thereto. For instance, a loading moiety may be attached to each end of the target polynucleotide. In such embodiments, one loading moiety is preferably a Y adaptor and the other loading moiety may be a bridging moiety, such as a hairpin loop adaptor. These are discussed in more detail below.

The one or more loading moieties may be attached to the target polynucleotide in any manner. The one or more loading moieties are preferably covalently attached to the target polynucleotide.

The one or more loading moieties are most preferably ligated to the target polynucleotide. The one or more loading moieties may be ligated to either end of the polynucleotide, i.e. the 5′ or the 3′ end. Loading moieties may be ligated to both ends of the target polynucleotide. The one or more loading moieties may be ligated to the polynucleotide using any method known in the art. The one or more loading moieties may be ligated to the polynucleotide in the absence of ATP or using gamma-S-ATP (ATPγS) instead of ATP.

The one or more loading moieties may be ligated using a ligase, such as T4 DNA ligase, E. coli DNA ligase, Taq DNA ligase, Tma DNA ligase and 9° N DNA ligase. The ligase is preferably used under the conditions set out in Example 3.

The method preferably further comprises removing the ligase from the method conditions.

The one or more polynucleotide binding proteins preferably remain bound (attached) to the loading moiety once the loading moiety has been attached to the target polynucleotide. After they have been attached in accordance with the invention, the one or more polynucleotide binding proteins may unbind from the one or more loading moieties.

Membrane

Any membrane may be used in accordance with the invention. Suitable membranes are well-known in the art. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, which have both hydrophilic and lipophilic properties. The amphiphilic molecules may be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles which form a monolayer are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymeric materials in which two or more monomer sub-units that are polymerized together to create a single polymer chain. Block copolymers typically have properties that are contributed by each monomer sub-unit. However, a block copolymer may have unique properties that polymers formed from the individual sub-units do not possess. Block copolymers can be engineered such that one of the monomer sub-units is hydrophobic (i.e. lipophilic), whilst the other sub-unit(s) are hydrophilic whilst in aqueous media. In this case, the block copolymer may possess amphiphilic properties and may form a structure that mimics a biological membrane. The block copolymer may be a diblock (consisting of two monomer sub-units), but may also be constructed from more than two monomer sub-units to form more complex arrangements that behave as amphiphiles. The copolymer may be a triblock, tetrablock or pentablock copolymer. The membrane is preferably a triblock copolymer membrane.

Archaebacterial bipolar tetraether lipids are naturally occurring lipids that are constructed such that the lipid forms a monolayer membrane. These lipids are generally found in extremophiles that survive in harsh biological environments, thermophiles, halophiles and acidophiles. Their stability is believed to derive from the fused nature of the final bilayer. It is straightforward to construct block copolymer materials that mimic these biological entities by creating a triblock polymer that has the general motif hydrophilic-hydrophobic-hydrophilic. This material may form monomeric membranes that behave similarly to lipid bilayers and encompass a range of phase behaviours from vesicles through to laminar membranes. Membranes formed from these triblock copolymers hold several advantages over biological lipid membranes. Because the triblock copolymer is synthesized, the exact construction can be carefully controlled to provide the correct chain lengths and properties required to form membranes and to interact with pores and other proteins.

Block copolymers may also be constructed from sub-units that are not classed as lipid sub-materials; for example a hydrophobic polymer may be made from siloxane or other non-hydrocarbon based monomers. The hydrophilic sub-section of block copolymer can also possess low protein binding properties, which allows the creation of a membrane that is highly resistant when exposed to raw biological samples. This head group unit may also be derived from non-classical lipid head-groups.

Triblock copolymer membranes also have increased mechanical and environmental stability compared with biological lipid membranes, for example a much higher operational temperature or pH range. The synthetic nature of the block copolymers provides a platform to customize polymer based membranes for a wide range of applications.

The membrane is most preferably one of the membranes disclosed in International Application No. PCT/GB2013/052766 or PCT/GB2013/052767.

The amphiphilic molecules may be chemically-modified or functionalised to facilitate coupling of the polynucleotide.

The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is typically planar. The amphiphilic layer may be curved. The amphiphilic layer may be supported.

Amphiphilic membranes are typically naturally mobile, essentially acting as two dimensional fluids with lipid diffusion rates of approximately 10⁻⁸ cm s−1. This means that the detector and coupled polynucleotide can typically move within an amphiphilic membrane.

The membrane may be a lipid bilayer. Lipid bilayers are models of cell membranes and serve as excellent platforms for a range of experimental studies. For example, lipid bilayers can be used for in vitro investigation of membrane proteins by single-channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer may be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, a planar lipid bilayer, a supported bilayer or a liposome. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in International Application No. PCT/GB08/000563 (published as WO 2008/102121), International Application No. PCT/GB08/004127 (published as WO 2009/077734) and International Application No. PCT/GB2006/001057 (published as WO 2006/100484).

Methods for forming lipid bilayers are known in the art. Suitable methods are disclosed in the Example. Lipid bilayers are commonly formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), in which a lipid monolayer is carried on aqueous solution/air interface past either side of an aperture which is perpendicular to that interface. The lipid is normally added to the surface of an aqueous electrolyte solution by first dissolving it in an organic solvent and then allowing a drop of the solvent to evaporate on the surface of the aqueous solution on either side of the aperture. Once the organic solvent has evaporated, the solution/air interfaces on either side of the aperture are physically moved up and down past the aperture until a bilayer is formed. Planar lipid bilayers may be formed across an aperture in a membrane or across an opening into a recess.

The method of Montal & Mueller is popular because it is a cost-effective and relatively straightforward method of forming good quality lipid bilayers that are suitable for protein pore insertion. Other common methods of bilayer formation include tip-dipping, painting bilayers and patch-clamping of liposome bilayers.

Tip-dipping bilayer formation entails touching the aperture surface (for example, a pipette tip) onto the surface of a test solution that is carrying a monolayer of lipid. Again, the lipid monolayer is first generated at the solution/air interface by allowing a drop of lipid dissolved in organic solvent to evaporate at the solution surface. The bilayer is then formed by the Langmuir-Schaefer process and requires mechanical automation to move the aperture relative to the solution surface.

For painted bilayers, a drop of lipid dissolved in organic solvent is applied directly to the aperture, which is submerged in an aqueous test solution. The lipid solution is spread thinly over the aperture using a paintbrush or an equivalent. Thinning of the solvent results in formation of a lipid bilayer. However, complete removal of the solvent from the bilayer is difficult and consequently the bilayer formed by this method is less stable and more prone to noise during electrochemical measurement.

Patch-clamping is commonly used in the study of biological cell membranes. The cell membrane is clamped to the end of a pipette by suction and a patch of the membrane becomes attached over the aperture. The method has been adapted for producing lipid bilayers by clamping liposomes which then burst to leave a lipid bilayer sealing over the aperture of the pipette. The method requires stable, giant and unilamellar liposomes and the fabrication of small apertures in materials having a glass surface.

Liposomes can be formed by sonication, extrusion or the Mozafari method (Colas et al. (2007) Micron 38:841-847).

In a preferred embodiment, the lipid bilayer is formed as described in International Application No. PCT/GB08/004127 (published as WO 2009/077734). Advantageously in this method, the lipid bilayer is formed from dried lipids. In a most preferred embodiment, the lipid bilayer is formed across an opening as described in WO2009/077734 (PCT/GB08/004127).

A lipid bilayer is formed from two opposing layers of lipids. The two layers of lipids are arranged such that their hydrophobic tail groups face towards each other to form a hydrophobic interior. The hydrophilic head groups of the lipids face outwards towards the aqueous environment on each side of the bilayer. The bilayer may be present in a number of lipid phases including, but not limited to, the liquid disordered phase (fluid lamellar), liquid ordered phase, solid ordered phase (lamellar gel phase, interdigitated gel phase) and planar bilayer crystals (lamellar sub-gel phase, lamellar crystalline phase).

Any lipid composition that forms a lipid bilayer may be used. The lipid composition is chosen such that a lipid bilayer having the required properties, such surface charge, ability to support membrane proteins, packing density or mechanical properties, is formed. The lipid composition can comprise one or more different lipids. For instance, the lipid composition can contain up to 100 lipids. The lipid composition preferably contains 1 to 10 lipids. The lipid composition may comprise naturally-occurring lipids and/or artificial lipids.

The lipids typically comprise a head group, an interfacial moiety and two hydrophobic tail groups which may be the same or different. Suitable head groups include, but are not limited to, neutral head groups, such as diacylglycerides (DG) and ceramides (CM); zwitterionic head groups, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM); negatively charged head groups, such as phosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol (PI), phosphatic acid (PA) and cardiolipin (CA); and positively charged headgroups, such as trimethylammonium-Propane (TAP). Suitable interfacial moieties include, but are not limited to, naturally-occurring interfacial moieties, such as glycerol-based or ceramide-based moieties. Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains, such as lauric acid (n-Dodecanolic acid), myristic acid (n-Tetradecononic acid), palmitic acid (n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and arachidic (n-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid (cis-9-Octadecanoic); and branched hydrocarbon chains, such as phytanoyl. The length of the chain and the position and number of the double bonds in the unsaturated hydrocarbon chains can vary. The length of the chains and the position and number of the branches, such as methyl groups, in the branched hydrocarbon chains can vary. The hydrophobic tail groups can be linked to the interfacial moiety as an ether or an ester. The lipids may be mycolic acid.

The lipids can also be chemically-modified. The head group or the tail group of the lipids may be chemically-modified. Suitable lipids whose head groups have been chemically-modified include, but are not limited to, PEG-modified lipids, such as 1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000]; functionalised PEG Lipids, such as 1,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N-[Biotinyl(Polyethylene Glycol)2000]; and lipids modified for conjugation, such as 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(succinyl) and 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(Biotinyl). Suitable lipids whose tail groups have been chemically-modified include, but are not limited to, polymerisable lipids, such as 1,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine; fluorinated lipids, such as 1-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3-Phosphocholine; deuterated lipids, such as 1,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine; and ether linked lipids, such as 1,2-Di-O-phytanyl-sn-Glycero-3-Phosphocholine. The lipids may be chemically-modified or functionalised to facilitate coupling of the polynucleotide.

The amphiphilic layer, for example the lipid composition, typically comprises one or more additives that will affect the properties of the layer. Suitable additives include, but are not limited to, fatty acids, such as palmitic acid, myristic acid and oleic acid; fatty alcohols, such as palmitic alcohol, myristic alcohol and oleic alcohol; sterols, such as cholesterol, ergosterol, lanosterol, sitosterol and stigmasterol; lysophospholipids, such as 1-Acyl-2-Hydroxy-sn-Glycero-3-Phosphocholine; and ceramides.

In another preferred embodiment, the membrane is a solid state layer. Solid state layers can be formed from both organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials such as Si₃N₄, Al₂O₃, and SiO, organic and inorganic polymers such as polyamide, plastics such as Teflon® or elastomers such as two-component addition-cure silicone rubber, and glasses. The solid state layer may be formed from graphene. Suitable graphene layers are disclosed in International Application No. PCT/US2008/010637 (published as WO 2009/035647).

The method is typically carried out using (i) an artificial amphiphilic layer comprising a pore, (ii) an isolated, naturally-occurring lipid bilayer comprising a pore, or (iii) a cell having a pore inserted therein. The method is typically carried out using an artificial amphiphilic layer, such as an artificial triblock copolymer layer. The layer may comprise other transmembrane and/or intramembrane proteins as well as other molecules in addition to the pore. Suitable apparatus and conditions are discussed below. The method of the invention is typically carried out in vitro.

Coupling

The one or more loading moieties preferably comprise one or more anchors that are capable of coupling to a membrane. Once attached in accordance with the invention, the one or more anchors are capable of coupling the target polynucleotide to a membrane.

In the characterisation method of the invention, the target polynucleotide is preferably coupled to the membrane using one or more anchors. The target polynucleotide may be coupled to the membrane using any known method.

Each anchor comprises a group which couples (or binds) to the one or more loading moieties and a group which couples (or binds) to the membrane. Each anchor may covalently couple (or bind) to the moiety and/or the membrane.

Each moiety may comprise any number of anchors, such as 2, 3, 4 or more anchors. For instance, one target polynucleotide may be coupled to the membrane using two anchors each of which separately couples (or binds) to both the polynucleotide via a moiety and membrane.

If the membrane is an amphiphilic layer, such as a copolymer membrane or a lipid bilayer, the one or more anchors preferably comprise a polypeptide anchor present in the membrane and/or a hydrophobic anchor present in the membrane. The hydrophobic anchor is preferably a lipid, fatty acid, sterol, carbon nanotube, polypeptide, protein or amino acid, for example cholesterol, palmitate or tocopherol. In preferred embodiments, the one or more anchors are not the detector.

The components of the membrane, such as the amphiphilic molecules, copolymer or lipids, may be chemically-modified or functionalised to form the one or more anchors. Examples of suitable chemical modifications and suitable ways of functionalising the components of the membrane are discussed in more detail below. Any proportion of the membrane components may be functionalized, for example at least 0.01%, at least 0.1%, at least 1%, at least 10%, at least 25%, at least 50% or 100%.

The one or more anchors used to couple the polynucleotide to the membrane preferably comprise a linker. The one or more anchors may comprise one or more, such as 2, 3, 4 or more, linkers. One linker may be used couple more than one, such as 2, 3, 4 or more, polynucleotides to the membrane.

Preferred linkers include, but are not limited to, polymers, such as polynucleotides, polyethylene glycols (PEGs), polysaccharides and polypeptides. These linkers may be linear, branched or circular. For instance, the linker may be a circular polynucleotide. The polynucleotide may hybridise to a complementary sequence on the circular polynucleotide linker.

The one or more anchors or one or more linkers may comprise a component that can be cut or broken down, such as a restriction site or a photolabile group.

Functionalised linkers and the ways in which they can couple molecules are known in the art. For instance, linkers functionalised with maleimide groups will react with and attach to cysteine residues in proteins. In the context of this invention, the protein may be present in the membrane or may be used to couple (or bind) to the one or more loading moieties. This is discussed in more detail below.

Crosslinkage of polynucleotides can be avoided using a “lock and key” arrangement. Only one end of each linker may react together to form a longer linker and the other ends of the linker each react with the loading moiety or membrane respectively. Such linkers are described in International Application No. PCT/GB10/000132 (published as WO 2010/086602).

The use of a linker is preferred in the sequencing embodiments discussed below. If a polynucleotide is permanently coupled directly to the membrane in the sense that it does not uncouple when interacting with the detector (i.e. does not uncouple in step (b) or (e)), then some sequence data will be lost as the sequencing run cannot continue to the end of the polynucleotide due to the distance between the membrane and the detector. If a linker is used, then the polynucleotide can be processed to completion.

The coupling may be permanent or stable. In other words, the coupling may be such that the polynucleotide remains coupled to the membrane when interacting with the pore.

The coupling may be transient. In other words, the coupling may be such that the polynucleotide may decouple from the membrane when interacting with the pore.

For certain applications, such as aptamer detection, the transient nature of the coupling is preferred. If a permanent or stable linker is attached directly to either the 5′ or 3′ end of a polynucleotide and the linker is shorter than the distance between the membrane and the transmembrane pore's channel, then some sequence data will be lost as the sequencing run cannot continue to the end of the polynucleotide. If the coupling is transient, then when the coupled end randomly becomes free of the membrane, then the polynucleotide can be processed to completion. Chemical groups that form permanent/stable or transient links are discussed in more detail below. The polynucleotide may be transiently coupled to an amphiphilic layer or triblock copolymer membrane using cholesterol or a fatty acyl chain. Any fatty acyl chain having a length of from 6 to 30 carbon atom, such as hexadecanoic acid, may be used.

In preferred embodiments, one or more anchors are capable of being coupled to an amphiphilic layer such as a triblock copolymer membrane or lipid bilayer. Coupling of nucleic acids to synthetic lipid bilayers has been carried out previously with various different tethering strategies. These are summarised in Table 1 below.

TABLE 1 Anchor Type of comprising coupling Reference Thiol Stable Yoshina-Ishii, C. and S. G. Boxer (2003). “Arrays of mobile tethered vesicles on supported lipid bilayers.” J Am Chem Soc 125(13): 3696-7. Biotin Stable Nikolov, V., R. Lipowsky, et al. (2007). “Behavior of giant vesicles with anchored DNA molecules.” Biophys J 92(12): 4356-68 Cholesterol Transient Pfeiffer, I. and F. Hook (2004). “Bivalent cholesterol-based coupling of oligonucletides to lipid membrane assemblies.” J Am Chem Soc 126(33): 10224-5 Surfactant Stable van Lengerich, B., R. J. Rawle, (e.g. Lipid, et al. “Covalent attachment of lipid Palmitate, etc) vesicles to a fluid-supported bilayer allows observation of DNA- mediated vesicle interactions.” Langmuir 26(11): 8666-72

Synthetic polynucleotides and/or linkers may be functionalised using a modified phosphoramidite in the synthesis reaction, which is easily compatible for the direct addition of suitable anchoring groups, such as cholesterol, tocopherol, palmitate, thiol, lipid and biotin groups. These different attachment chemistries give a suite of options for attachment to loading moieties. Each different modification group couples the loading moiety in a slightly different way and coupling is not always permanent so giving different dwell times for the polynucleotide to the membrane. The advantages of transient coupling are discussed above.

Coupling of polynucleotides to a linker or to a functionalised membrane can also be achieved by a number of other means provided that a complementary reactive group or an anchoring group can be added to the polynucleotide loading moiety. The addition of reactive groups to either end of a polynucleotide has been reported previously. A thiol group can be added to the 5′ of ssDNA or dsDNA using T4 polynucleotide kinase and ATPγS (Grant, G. P. and P. Z. Qin (2007). “A facile method for attaching nitroxide spin labels at the 5′ terminus of nucleic acids.” Nucleic Acids Res 35(10): e77). An azide group can be added to the 5′-phosphate of ssDNA or dsDNA using T4 polynucleotide kinase and γ-[2-Azidoethyl]-ATP or γ-[6-Azidohexyl]-ATP. Using thiol or Click chemistry a tether, containing either a thiol, iodoacetamide OPSS or maleimide group (reactive to thiols) or a DIBO (dibenzocyclooxtyne) or alkyne group (reactive to azides), can be covalently attached to the polynucleotide loading moiety. A more diverse selection of chemical groups, such as biotin, thiols and fluorophores, can be added using terminal transferase to incorporate modified oligonucleotides to the 3′ of ssDNA (Kumar, A., P. Tchen, et al. (1988). “Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase.” Anal Biochem 169(2): 376-82). Streptavidin/biotin and/or streptavidin/desthiobiotin coupling may be used for any other polynucleotide loading moiety. It may also be possible that anchors may be directly added to polynucleotide loading moieties using terminal transferase with suitably modified nucleotides (e.g. cholesterol or palmitate).

The one or more anchors are preferably capable of coupling the loading moiety/polynucleotide to the membrane via hybridisation. Hybridisation in the one or more anchors allows coupling in a transient manner as discussed above. The hybridisation may be present in any part of the one or more anchors, such as between the one or more anchors and the loading moiety, within the one or more anchors or between the one or more anchors and the membrane. For instance, a linker may comprise two or more polynucleotides, such as 3, 4 or 5 polynucleotides, hybridised together. The one or more anchors may hybridise to the polynucleotide loading moiety. The one or more anchors may hybridise directly to the polynucleotide loading moiety such as directly to a Y adaptor and/or leader sequence or directly to a bridging moiety adaptor, such as a hairpin loop adaptor (as discussed below). Alternatively, the one or more anchors may be hybridised to one or more, such as 2 or 3, intermediate polynucleotides (or “splints”) which are hybridised to the polynucleotide loading moiety, to a Y adaptor and/or leader sequence or to a bridging moiety adaptor attached to the polynucleotide (as discussed below).

The one or more anchors may comprise a single stranded or double stranded polynucleotide. One part of the anchor may be ligated to a single stranded or double stranded polynucleotide loading moiety. Ligation of short pieces of ssDNA have been reported using T4 RNA ligase I (Troutt, A. B., M. G. McHeyzer-Williams, et al. (1992). “Ligation-anchored PCR: a simple amplification technique with single-sided specificity.” Proc Natl Acad Sci USA 89(20): 9823-5). Alternatively, either a single stranded or double stranded polynucleotide can be ligated to a double stranded polynucleotide and then the two strands separated by thermal or chemical denaturation. To a double stranded polynucleotide, it is possible to add either a piece of single stranded polynucleotide to one or both of the ends of the duplex, or a double stranded polynucleotide to one or both ends. For addition of single stranded polynucleotides to the a double stranded polynucleotide, this can be achieved using T4 RNA ligase I as for ligation to other regions of single stranded polynucleotides. For addition of double stranded polynucleotides to a double stranded polynucleotide then ligation can be “blunt-ended”, with complementary 3′ dA/dT tails on the polynucleotide and added polynucleotide respectively (as is routinely done for many sample prep applications to prevent concatemer or dimer formation) or using “sticky-ends” generated by restriction digestion of the polynucleotide and ligation of compatible adapters. Then, when the duplex is melted, each single strand will have either a 5′ or 3′ modification if a single stranded polynucleotide was used for ligation or a modification at the 5′ end, the 3′ end or both if a double stranded polynucleotide was used for ligation.

If the loading moiety is synthetic, the one or more anchors can be incorporated during its chemical synthesis. For instance, a polynucleotide loading moiety can be synthesised using a primer having a reactive group attached to it.

Adenylated polynucleotides are intermediates in ligation reactions, where an adenosine-monophosphate is attached to the 5′-phosphate of the polynucleotide. Various kits are available for generation of this intermediate, such as the 5′ DNA Adenylation Kit from NEB. By substituting ATP in the reaction for a modified nucleotide triphosphate, then addition of reactive groups (such as thiols, amines, biotin, azides, etc) to the 5′ of a polynucleotide can be possible. It may also be possible that anchors could be directly added to polynucleotide loading moieties using a 5′ DNA adenylation kit with suitably modified nucleotides (e.g. cholesterol or palmitate).

A common technique for the amplification of sections of genomic DNA is using polymerase chain reaction (PCR). Here, using two synthetic oligonucleotide primers, a number of copies of the same section of DNA can be generated, where for each copy the 5′ of each strand in the duplex will be a synthetic polynucleotide. Single or multiple nucleotides can be added to 3′ end of single or double stranded DNA by employing a polymerase. Examples of polymerases which could be used include, but are not limited to, Terminal Transferase, Klenow and E. coli Poly(A) polymerase. By substituting ATP in the reaction for a modified nucleotide triphosphate then anchors, such as a cholesterol, thiol, amine, azide, biotin or lipid, can be incorporated into double stranded polynucleotide loading moieties. Therefore, each copy of the amplified polynucleotide loading moiety will contain an anchor.

Ideally, the loading moiety/polynucleotide is coupled to the membrane without having to functionalise the loading moiety/polynucleotide. This can be achieved by coupling the one or more anchors, such as a polynucleotide binding protein or a chemical group, to the membrane and allowing the one or more anchors to interact with the loading moiety/polynucleotide or by functionalizing the membrane. The one or more anchors may be coupled to the membrane by any of the methods described herein. In particular, the one or more anchors may comprise one or more linkers, such as maleimide functionalised linkers.

The one or more anchors can comprise any group that couples to, binds to or interacts with single or double stranded polynucleotides, specific nucleotide sequences within the loading moiety or patterns of modified nucleotides within the loading moiety, or any other ligand that is present on the loading moiety.

Suitable binding proteins for use in anchors include, but are not limited to, E. coli single stranded binding protein, P5 single stranded binding protein, T4 gp32 single stranded binding protein, the TOPO V dsDNA binding region, human histone proteins, E. coli HU DNA binding protein and other archaeal, prokaryotic or eukaryotic single stranded or double stranded polynucleotide (or nucleic acid) binding proteins, including those listed below.

The specific nucleotide sequences in the loading moiety could be sequences recognised by transcription factors, ribosomes, endonucleases, topoisomerases or replication initiation factors. The patterns of modified nucleotides could be patterns of methylation or damage.

The one or more anchors can comprise any group which couples to, binds to, intercalates with or interacts with a polynucleotide loading moiety. The group may intercalate or interact with the polynucleotide via electrostatic, hydrogen bonding or Van der Waals interactions. Such groups include a lysine monomer, poly-lysine (which will interact with ssDNA or dsDNA), ethidium bromide (which will intercalate with dsDNA), universal bases or universal nucleotides (which can hybridise with any polynucleotide) and osmium complexes (which can react to methylated bases). A polynucleotide loading moiety may therefore be coupled to the membrane using one or more universal nucleotides attached to the membrane. Each universal nucleotide may be coupled to the membrane using one or more linkers. The universal nucleotide preferably comprises one of the following nucleobases: hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, formylindole, 3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole or phenyl (C6-aromatic ring). The universal nucleotide more preferably comprises one of the following nucleosides: 2′-deoxyinosine, inosine, 7-deaza-2′-deoxyinosine, 7-deaza-inosine, 2-aza-deoxyinosine, 2-aza-inosine, 2-0′-methylinosine, 4-nitroindole 2′-deoxyribonucleoside, 4-nitroindole ribonucleoside, 5-nitroindole 2′-deoxyribonucleoside, 5-nitroindole ribonucleoside, 6-nitroindole 2′-deoxyribonucleoside, 6-nitroindole ribonucleoside, 3-nitropyrrole 2′-deoxyribonucleoside, 3-nitropyrrole ribonucleoside, an acyclic sugar analogue of hypoxanthine, nitroimidazole 2′-deoxyribonucleoside, nitroimidazole ribonucleoside, 4-nitropyrazole 2′-deoxyribonucleoside, 4-nitropyrazole ribonucleoside, 4-nitrobenzimidazole 2′-deoxyribonucleoside, 4-nitrobenzimidazole ribonucleoside, 5-nitroindazole 2′-deoxyribonucleoside, 5-nitroindazole ribonucleoside, 4-aminobenzimidazole 2′-deoxyribonucleoside, 4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside, phenyl C-2′-deoxyribosyl nucleoside, 2′-deoxynebularine, 2′-deoxyisoguanosine, K-T-deoxyribose, P-2′-deoxyribose and pyrrolidine. The universal nucleotide more preferably comprises 2′-deoxyinosine. The universal nucleotide is more preferably IMP or dIMP. The universal nucleotide is most preferably dPMP (2′-Deoxy-P-nucleoside monophosphate) or dKMP (N6-methoxy-2, 6-diaminopurine monophosphate).

The one or more anchors may couple to (or bind to) the polynucleotide loading moiety via Hoogsteen hydrogen bonds (where two nucleobases are held together by hydrogen bonds) or reversed Hoogsteen hydrogen bonds (where one nucleobase is rotated through 180° with respect to the other nucleobase). For instance, the one or more anchors may comprise one or more nucleotides, one or more oligonucleotides or one or more polynucleotides which form Hoogsteen hydrogen bonds or reversed Hoogsteen hydrogen bonds with the polynucleotide loading moiety. These types of hydrogen bonds allow a third polynucleotide strand to wind around a double stranded helix and form a triplex. The one or more anchors may couple to (or bind to) a double stranded polynucleotide loading moiety by forming a triplex with the double stranded duplex.

In this embodiment at least 1%, at least 10%, at least 25%, at least 50% or 100% of the membrane components may be functionalized.

Where the one or more anchors comprise a protein, they may be able to anchor directly into the membrane without further functonalisation, for example if it already has an external hydrophobic region which is compatible with the membrane. Examples of such proteins include, but are not limited to, transmembrane proteins, intramembrane proteins and membrane proteins. Alternatively the protein may be expressed with a genetically fused hydrophobic region which is compatible with the membrane. Such hydrophobic protein regions are known in the art.

The one or more anchors are preferably mixed with the one or more loading moieties before contacting with the membrane, but the one or more anchors may be contacted with the membrane and subsequently contacted with the one or more loading moieties.

In another aspect a loading moiety may be functionalised, using methods described above, so that it can be recognised by a specific binding group. Specifically the loading moiety may be functionalised with a ligand such as biotin (for binding to streptavidin), amylose (for binding to maltose binding protein or a fusion protein), Ni-NTA (for binding to poly-histidine or poly-histidine tagged proteins) or a peptides (such as an antigen).

According to a preferred embodiment, the one or more anchors may be used to couple a polynucleotide to the membrane when the polynucleotide is attached to a loading moiety comprising a leader sequence which preferentially threads into the pore. Leader sequences are discussed in more detail below. Preferably, the polynucleotide is attached (such as ligated) to a leader sequence which preferentially threads into the pore. Such a leader sequence may comprise a homopolymeric polynucleotide or an abasic region. The leader sequence is typically designed to hybridise to the one or more anchors either directly or via one or more intermediate polynucleotides (or splints). In such instances, the one or more anchors typically comprise a polynucleotide sequence which is complementary to a sequence in the leader sequence or a sequence in the one or more intermediate polynucleotides (or splints). In such instances, the one or more splints typically comprise a polynucleotide sequence which is complementary to a sequence in the leader sequence.

An example of a molecule used in chemical attachment is EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride). Reactive groups can also be added to the 5′ of polynucleotides using commercially available kits (Thermo Pierce, Part No. 22980). Suitable methods include, but are not limited to, transient affinity attachment using histidine residues and Ni-NTA, as well as more robust covalent attachment by reactive cysteines, lysines or non natural amino acids.

Transmembrane Pore

The characterisation method of the invention comprises taking one or more measurements as the target polynucleotide moves with respect to the transmembrane pore. A variety of different types of measurements may be made using the pore. This includes without limitation: electrical measurements and optical measurements. Possible electrical measurements include: current measurements, impedance measurements, tunnelling measurements (Ivanov A P et al., Nano Lett. 2011 Jan. 12; 11(1):279-85), and FET measurements (International Application WO 2005/124888). Optical measurements may be combined with electrical measurements (Soni G V et al., Rev Sci Instrum. 2010 January; 81(1):014301). The measurement may be a transmembrane current measurement such as measurement of ionic current flowing through the pore.

Electrical measurements may be made using standard single channel recording equipment as describe in Stoddart D et al., Proc Natl Acad Sci, 12;106(19):7702-7, Lieberman K R et al, J Am Chem Soc. 2010; 132(50):17961-72, and International Application WO 2000/28312. Alternatively, electrical measurements may be made using a multi-channel system, for example as described in International Application WO 2009/077734 and International Application WO 2011/067559.

The method is preferably carried out with a potential applied across the membrane. The applied potential may be a voltage potential. Alternatively, the applied potential may be a chemical potential. An example of this is using a salt gradient across a membrane, such as an amphiphilic layer. A salt gradient is disclosed in Holden et al., J Am Chem Soc. 2007 Jul. 11; 129(27):8650-5. In some instances, the current passing through the detector (or pore) as a polynucleotide moves with respect to the pore is used to estimate or determine the sequence of the polynucleotide. This is strand sequencing.

The method comprises contacting the target polynucleotide with a transmembrane pore. A transmembrane pore is a structure that crosses the membrane to some degree. It permits hydrated ions driven by an applied potential to flow across or within the membrane. The transmembrane pore typically crosses the entire membrane so that hydrated ions may flow from one side of the membrane to the other side of the membrane. However, the transmembrane pore does not have to cross the membrane. It may be closed at one end. For instance, the pore may be a well, gap, channel, trench or slit in the membrane along which or into which hydrated ions may flow.

Any transmembrane pore may be used in the invention. The pore may be biological or artificial. Suitable pores include, but are not limited to, protein pores, polynucleotide pores and solid state pores. The pore may be a DNA origami pore (Langecker et al., Science, 2012; 338: 932-936).

The transmembrane pore is preferably a transmembrane protein pore. A transmembrane protein pore is a polypeptide or a collection of polypeptides that permits hydrated ions, such as analyte, to flow from one side of a membrane to the other side of the membrane. In the present invention, the transmembrane protein pore is capable of forming a pore that permits hydrated ions driven by an applied potential to flow from one side of the membrane to the other. The transmembrane protein pore preferably permits analyte such as nucleotides to flow from one side of the membrane, such as a triblock copolymer membrane, to the other. The transmembrane protein pore allows a polynucleotide, such as DNA or RNA, to be moved with respect to the pore, such as through the pore.

The transmembrane protein pore may be a monomer or an oligomer. The pore is preferably made up of several repeating subunits, such as at least 6, at least 7, at least 8 or at least 9 subunits. The pore is preferably a hexameric, heptameric, octameric or nonameric pore. The pore may be a homo-oligomer or a hetero-oligomer.

The transmembrane protein pore typically comprises a barrel or channel through which the ions may flow. The subunits of the pore typically surround a central axis and contribute strands to a transmembrane β barrel or channel or a transmembrane α-helix bundle or channel. The barrel or channel of the transmembrane protein pore typically comprises amino acids that facilitate interaction with analyte, such as nucleotides, polynucleotides or nucleic acids. These amino acids are preferably located near a constriction of the barrel or channel. The transmembrane protein pore typically comprises one or more positively charged amino acids, such as arginine, lysine or histidine, or aromatic amino acids, such as tyrosine or tryptophan. These amino acids typically facilitate the interaction between the pore and nucleotides, polynucleotides or nucleic acids.

Transmembrane protein pores for use in accordance with the invention can be derived from β-barrel pores or α-helix bundle pores. β-barrel pores comprise a barrel or channel that is formed from β-strands. Suitable β-barrel pores include, but are not limited to, β-toxins, such as α-hemolysin, anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and Neisseria autotransporter lipoprotein (NalP) and other pores such as lysenin. α-helix bundle pores comprise a barrel or channel that is formed from α-helices. Suitable α-helix bundle pores include, but are not limited to, inner membrane proteins and a outer membrane proteins, such as WZA and ClyA toxin. The transmembrane pore may be derived from lysenin. Suitable pores derived from lysenin are disclosed in International Application No. PCT/GB2013/050667 (published as WO 2013/153359). The transmembrane pore may be derived from Msp or from α-hemolysin (α-HL).

The transmembrane protein pore is preferably derived from Msp, preferably from MspA. Such a pore will be oligomeric and typically comprises 7, 8, 9 or 10 monomers derived from Msp. The pore may be a homo-oligomeric pore derived from Msp comprising identical monomers. Alternatively, the pore may be a hetero-oligomeric pore derived from Msp comprising at least one monomer that differs from the others. Preferably the pore is derived from MspA or a homolog or paralog thereof.

A monomer derived from Msp typically comprises the sequence shown in SEQ ID NO: 2 or a variant thereof. SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspA monomer. It includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K. A variant of SEQ ID NO: 2 is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 2 and which retains its ability to form a pore. The ability of a variant to form a pore can be assayed using any method known in the art. For instance, the variant may be inserted into an amphiphilic layer along with other appropriate subunits and its ability to oligomerise to form a pore may be determined. Methods are known in the art for inserting subunits into membranes, such as amphiphilic layers. For example, subunits may be suspended in a purified form in a solution containing a triblock copolymer membrane such that it diffuses to the membrane and is inserted by binding to the membrane and assembling into a functional state. Alternatively, subunits may be directly inserted into the membrane using the “pick and place” method described in M. A. Holden, H. Bayley. J. Am. Chem. Soc. 2005, 127, 6502-6503 and International Application No. PCT/GB2006/001057 (published as WO 2006/100484).

Over the entire length of the amino acid sequence of SEQ ID NO: 2, a variant will preferably be at least 50% homologous to that sequence based on amino acid identity. More preferably, the variant may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 2 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous amino acids (“hard homology”).

Standard methods in the art may be used to determine homology. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspA monomer. The variant may comprise any of the mutations in the MspB, C or D monomers compared with MspA. The mature forms of MspB, C and D are shown in SEQ ID NOs: 5 to 7. In particular, the variant may comprise the following substitution present in MspB: A138P. The variant may comprise one or more of the following substitutions present in MspC: A96G, N102E and A138P. The variant may comprise one or more of the following mutations present in MspD: Deletion of G1, L2V, ESQ, L8V, D13G, W21A, D22E, K47T, I49H, I68V, D91G, A96Q, N102D, S103T, V1041, S136K and G141A. The variant may comprise combinations of one or more of the mutations and substitutions from Msp B, C and D. The variant preferably comprises the mutation L88N. A variant of SEQ ID NO: 2 has the mutation L88N in addition to all the mutations of MS-B1 and is called MS-(B2)8. The pore used in the invention is preferably MS-(B2)8. A variant of SEQ ID NO: 2 has the mutations G75S/G77S/L88N/Q126R in addition to all the mutations of MS-B1 and is called MS-B2C. The pore used in the invention is preferably MS-(B2)8 or MS-(B2C)8.

Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 2 in addition to those discussed above, for example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table 2 below. Where amino acids have similar polarity, this can also be determined by reference to the hydropathy scale for amino acid side chains in Table 3.

TABLE 2 Chemical properties of amino acids Ala aliphatic, hydrophobic, neutral Cys polar, hydrophobic, neutral Asp polar, hydrophilic, charged (−) Glu polar, hydrophilic, charged (−) Phe aromatic, hydrophobic, neutral Gly aliphatic, neutral His aromatic, polar, hydrophilic, charged (+) Ile aliphatic, hydrophobic, neutral Lys polar, hydrophilic, charged(+) Leu aliphatic, hydrophobic, neutral Met hydrophobic, neutral Asn polar, hydrophilic, neutral Pro hydrophobic, neutral Gln polar, hydrophilic, neutral Arg polar, hydrophilic, charged (+) Ser polar, hydrophilic, neutral Thr polar, hydrophilic, neutral Val aliphatic, hydrophobic, neutral Trp aromatic, hydrophobic, neutral Tyr aromatic, polar, hydrophobic

TABLE 3 Hydropathy scale Side Chain Hydropathy Ile 4.5 Val 4.2 Leu 3.8 Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly −0.4 Thr −0.7 Ser −0.8 Trp −0.9 Tyr −1.3 Pro −1.6 His −3.2 Glu −3.5 Gln −3.5 Asp −3.5 Asn −3.5 Lys −3.9 Arg −4.5

One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 may additionally be deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may include fragments of SEQ ID NO: 2. Such fragments retain pore forming activity. Fragments may be at least 50, 100, 150 or 200 amino acids in length. Such fragments may be used to produce the pores. A fragment preferably comprises the pore forming domain of SEQ ID NO: 2. Fragments must include one of residues 88, 90, 91, 105, 118 and 134 of SEQ ID NO: 2. Typically, fragments include all of residues 88, 90, 91, 105, 118 and 134 of SEQ ID NO: 2.

One or more amino acids may be alternatively or additionally added to the polypeptides described above. An extension may be provided at the amino terminal or carboxy terminal of the amino acid sequence of SEQ ID NO: 2 or polypeptide variant or fragment thereof. The extension may be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids. A carrier protein may be fused to an amino acid sequence according to the invention. Other fusion proteins are discussed in more detail below.

As discussed above, a variant is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 2 and which retains its ability to form a pore. A variant typically contains the regions of SEQ ID NO: 2 that are responsible for pore formation. The pore forming ability of Msp, which contains a β-barrel, is provided by β-sheets in each subunit. A variant of SEQ ID NO: 2 typically comprises the regions in SEQ ID NO: 2 that form β-sheets. One or more modifications can be made to the regions of SEQ ID NO: 2 that form β-sheets as long as the resulting variant retains its ability to form a pore. A variant of SEQ ID NO: 2 preferably includes one or more modifications, such as substitutions, additions or deletions, within its α-helices and/or loop regions.

The monomers derived from Msp may be modified to assist their identification or purification, for example by the addition of histidine residues (a hist tag), aspartic acid residues (an asp tag), a streptavidin tag or a flag tag, or by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered position on the pore. An example of this would be to react a gel-shift reagent to a cysteine engineered on the outside of the pore. This has been demonstrated as a method for separating hemolysin hetero-oligomers (Chem Biol. 1997 July; 4(7):497-505).

The monomer derived from Msp may be labelled with a revealing label. The revealing label may be any suitable label which allows the pore to be detected. Suitable labels are described below.

The monomer derived from Msp may also be produced using D-amino acids. For instance, the monomer derived from Msp may comprise a mixture of L-amino acids and D-amino acids. This is conventional in the art for producing such proteins or peptides.

The monomer derived from Msp contains one or more specific modifications to facilitate nucleotide discrimination. The monomer derived from Msp may also contain other non-specific modifications as long as they do not interfere with pore formation. A number of non-specific side chain modifications are known in the art and may be made to the side chains of the monomer derived from Msp. Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH₄, amidination with methylacetimidate or acylation with acetic anhydride.

The monomer derived from Msp can be produced using standard methods known in the art. The monomer derived from Msp may be made synthetically or by recombinant means. For example, the pore may be synthesized by in vitro translation and transcription (IVTT). Suitable methods for producing pores are discussed in International Application Nos. PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679 (published as WO 2010/004265) or PCT/GB10/000133 (published as WO 2010/086603). Methods for inserting pores into membranes are discussed.

The transmembrane protein pore is also preferably derived from α-hemolysin (α-HL). The wild type α-HL pore is formed of seven identical monomers or subunits (i.e. it is heptameric). The sequence of one monomer or subunit of α-hemolysin-NN is shown in SEQ ID NO: 4.

In some embodiments, the transmembrane protein pore is chemically modified. The pore can be chemically modified in any way and at any site. The transmembrane protein pore is preferably chemically modified by attachment of a molecule to one or more cysteines (cysteine linkage), attachment of a molecule to one or more lysines, attachment of a molecule to one or more non-natural amino acids, enzyme modification of an epitope or modification of a terminus. Suitable methods for carrying out such modifications are well-known in the art. The transmembrane protein pore may be chemically modified by the attachment of any molecule. For instance, the pore may be chemically modified by attachment of a dye or a fluorophore.

Any number of the monomers in the pore may be chemically modified. One or more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the monomers is preferably chemically modified as discussed above.

The reactivity of cysteine residues may be enhanced by modification of the adjacent residues. For instance, the basic groups of flanking arginine, histidine or lysine residues will change the pKa of the cysteines thiol group to that of the more reactive S⁻ group. The reactivity of cysteine residues may be protected by thiol protective groups such as dTNB. These may be reacted with one or more cysteine residues of the pore before a linker is attached.

The molecule (with which the pore is chemically modified) may be attached directly to the pore or attached via a linker as disclosed in International Application Nos. PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679 (published as WO 2010/004265) or PCT/GB10/000133 (published as WO 2010/086603).

Any of the proteins described herein, such as the transmembrane protein pores, may be modified to assist their identification or purification, for example by the addition of histidine residues (a his tag), aspartic acid residues (an asp tag), a streptavidin tag, a flag tag, a SUMO tag, a GST tag or a MBP tag, or by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered position on the pore or construct. An example of this would be to react a gel-shift reagent to a cysteine engineered on the outside of the pore. This has been demonstrated as a method for separating hemolysin hetero-oligomers (Chem Biol. 1997 July; 4(7):497-505).

The pore may be labelled with a revealing label. The revealing label may be any suitable label which allows the pore to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. ¹²⁵I, ³⁵S, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin.

Any of the proteins described herein, such as the transmembrane protein pores, may be made synthetically or by recombinant means. For example, the pore may be synthesized by in vitro translation and transcription (IVTT). The amino acid sequence of the pore may be modified to include non-naturally occurring amino acids or to increase the stability of the protein. When a protein is produced by synthetic means, such amino acids may be introduced during production. The pore may also be altered following either synthetic or recombinant production.

The pore may also be produced using D-amino acids. For instance, the pore or construct may comprise a mixture of L-amino acids and D-amino acids. This is conventional in the art for producing such proteins or peptides.

The pore may also contain other non-specific modifications as long as they do not interfere with pore formation or construct function. A number of non-specific side chain modifications are known in the art and may be made to the side chains of the protein(s). Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH₄, amidination with methylacetimidate or acylation with acetic anhydride.

Any of the proteins described herein, such as the transmembrane protein pores, can be produced using standard methods known in the art. Polynucleotide sequences encoding a pore or construct may be derived and replicated using standard methods in the art. Polynucleotide sequences encoding a pore or construct may be expressed in a bacterial host cell using standard techniques in the art. The pore may be produced in a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide. These methods are described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The pore may be produced in large scale following purification by any protein liquid chromatography system from protein producing organisms or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system and the Gilson HPLC system.

Spacers

If the one or more polynucleotide binding proteins are helicases and the one or more loading moieties comprise a polynucleotide, the one or more helicases may be stalled at one or more spacers as discussed in International Application No. PCT/GB2014/050175 (published as WO 2014/135838). Any configuration of one or more helicases and one or more spacers disclosed in the International Application may be used in this invention.

When a part of the target polynucleotide enters the pore and moves with respect to the pore, such as through the pore, along the field resulting from the applied potential, the one or more helicases are moved past the spacer by the pore as the polynucleotide moves with respect to the pore, such as through the pore. This is because the polynucleotide (including the one or more spacers) moves with respect to the pore, such as through the pore, and the one or more helicases remain on top of the pore.

The one or more spacers are preferably part of the loading moiety polynucleotide, for instance it/they interrupt(s) the polynucleotide sequence. The one or more spacers are preferably not part of one or more blocking molecules, such as speed bumps, hybridised to the polynucleotide.

There may be any number of spacers in the loading moiety polynucleotide, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more spacers. There are preferably two, four or six spacers in the polynucleotide. There may be spacer in different loading moiety polynucleotides, such as a spacer in the leader sequence and a spacer in the bridging moiety or the hairpin loop.

The one or more spacers each provides an energy barrier which the one or more helicases cannot overcome even in the active mode. The one or more spacers may stall the one or more helicases by reducing the traction of the helicase (for instance by removing the bases from the nucleotides in the polynucleotide) or physically blocking movement of the one or more helicases (for instance using a bulky chemical group).

The one or more spacers may comprise any molecule or combination of molecules that stalls the one or more helicases. The one or more spacers may comprise any molecule or combination of molecules that prevents the one or more helicases from moving along the polynucleotide. It is straightforward to determine whether or not the one or more helicases are stalled at one or more spacers in the absence of a transmembrane pore and an applied potential. For instance, this can be assayed as shown in the Examples, for instance the ability of a helicase to move past a spacer and displace a complementary strand of DNA can be measured by PAGE.

The one or more spacers typically comprise a linear molecule, such as a polymer. The one or more spacers typically have a different structure from the polynucleotide. For instance, if the polynucleotide is DNA, the one or more spacers are typically not DNA. In particular, if the polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the one or more spacers preferably comprise peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), bridged nucleic acid (BNA) or a synthetic polymer with nucleotide side chains. The one or more spacers may comprise one or more nucleotides in the opposite direction from the polynucleotide. For instance, the one or more spacers may comprise one or more nucleotides in the 3′ to 5′ direction when the polynucleotide is in the 5′ to 3′ direction. The nucleotides may be any of those discussed above.

The one or more spacers preferably comprises one or more nitroindoles, such as one or more 5-nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dTs), one or more inverted dideoxy-thymidines (ddTs), one or more dideoxy-cytidines (ddCs), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2′-O-Methyl RNA bases, one or more Iso-deoxycytidines (Iso-dCs), one or more Iso-deoxyguanosines (Iso-dGs), one or more iSpC3 groups (i.e. nucleotides which lack sugar and a base), one or more photo-cleavable (PC) groups, one or more hexandiol groups, one or more spacer 9 (iSp9) groups, one or more spacer 18 (iSp18) groups, a polymer or one or more thiol connections. The one or more spacers may comprise any combination of these groups. Many of these groups are commercially available from IDT® (Integrated DNA Technologies®).

The one or more spacers may contain any number of these groups. For instance, for 2-aminopurines, 2-6-diaminopurines, 5-bromo-deoxyuridines, inverted dTs, ddTs, ddCs, 5-methylcytidines, 5-hydroxymethylcytidines, 2′-O-Methyl RNA bases, Iso-dCs, Iso-dGs, iSpC3 groups, PC groups, hexandiol groups and thiol connections, the one or more spacers preferably comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. The one or more spacers preferably comprise 2, 3, 4, 5, 6, 7, 8 or more iSp9 groups. The one or more spacers preferably comprise 2, 3, 4, 5 or 6 or more iSp18 groups. The most preferred spacer is four iSp18 groups.

The polymer is preferably a polypeptide or a polyethylene glycol (PEG). The polypeptide preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids. The PEG preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more monomer units.

The one or more spacers preferably comprise one or more abasic nucleotides (i.e. nucleotides lacking a nucleobase), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more abasic nucleotides. The nucleobase can be replaced by —H (idSp) or —OH in the abasic nucleotide. Abasic spacers can be inserted into polynucleotides by removing the nucleobases from one or more adjacent nucleotides. For instance, polynucleotides may be modified to include 3-methyladenine, 7-methylguanine, 1,N6-ethenoadenine inosine or hypoxanthine and the nucleobases may be removed from these nucleotides using Human Alkyladenine DNA Glycosylase (hAAG). Alternatively, polynucleotides may be modified to include uracil and the nucleobases removed with Uracil-DNA Glycosylase (UDG). In one embodiment, the one or more spacers do not comprise any abasic nucleotides.

The one or more helicases may be stalled by (i.e. before) or on each linear molecule spacer. If linear molecule spacers are used, the loading moiety polynucleotide is preferably provided with a double stranded region of polynucleotide adjacent to the end of each spacer past which the one or more helicases are to be moved. The double stranded region typically helps to stall the one or more helicases on the adjacent spacer. The presence of the double stranded region(s) is particularly preferred if the method is carried out at a salt concentration of about 100 mM or lower. Each double stranded region is typically at least 10, such as at least 12, nucleotides in length. If the loading moiety polynucleotide used in the invention is single stranded, a double stranded region may formed by hybridising a shorter polynucleotide to a region adjacent to a spacer. The shorter polynucleotide is typically formed from the same nucleotides as the loading moiety polynucleotide, but may be formed from different nucleotides. For instance, the shorter polynucleotide may be formed from LNA or BNA.

If linear molecule spacers are used, the loading moiety polynucleotide is preferably provided with a blocking molecule at the end of each spacer opposite to the end past which the one or more helicases are to be moved. This can help to ensure that the one or more helicases remain stalled on each spacer. It may also help retain the one or more helicases on the polynucleotide in the case that it/they diffuse(s) off in solution. The blocking molecule may be any of the chemical groups discussed below which physically cause the one or more helicases to stall. The blocking molecule may be a double stranded region of polynucleotide.

The one or more spacers preferably comprise one or more chemical groups which physically cause the one or more helicases to stall. The one or more chemical groups are preferably one or more pendant chemical groups. The one or more chemical groups may be attached to one or more nucleobases in the polynucleotide. The one or more chemical groups may be attached to the polynucleotide backbone. Any number of these chemical groups may be present, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. Suitable groups include, but are not limited to, fluorophores, streptavidin and/or biotin, cholesterol, methylene blue, dinitrophenols (DNPs), digoxigenin and/or anti-digoxigenin and dibenzylcyclooctyne groups.

Different spacers in the loading moiety polynucleotide may comprise different stalling molecules. For instance, one spacer may comprise one of the linear molecules discussed above and another spacer may comprise one or more chemical groups which physically cause the one or more helicases to stall. A spacer may comprise any of the linear molecules discussed above and one or more chemical groups which physically cause the one or more helicases to stall, such as one or more abasics and a fluorophore.

Suitable spacers can be designed depending on the type of loading moiety polynucleotide and the conditions under which the method of the invention is carried out. Most helicases bind and move along DNA and so may be stalled using anything that is not DNA. Suitable molecules are discussed above.

The characterisation method of the invention is preferably carried out in the presence of free nucleotides and/or the presence of a helicase cofactor. This is discussed in more detail below. In the absence of the transmembrane pore and an applied potential, the one or more spacers are preferably capable of stalling the one or more helicases in the presence of free nucleotides and/or the presence of a helicase cofactor.

If the characterization method of the invention is carried out in the presence of free nucleotides and a helicase cofactor as discussed below (such that the one of more helicases are in the active mode), one or more longer spacers are typically used to ensure that the one or more helicases are stalled on the polynucleotide before they are contacted with the transmembrane pore and a potential is applied. One or more shorter spacers may be used in the absence of free nucleotides and a helicase cofactor (such that the one or more helicases are in the inactive mode).

The salt concentration also affects the ability of the one or more spacers to stall the one or more helicases. In the absence of the transmembrane pore and an applied potential, the one or more spacers are preferably capable of stalling the one or more helicases at a salt concentration of about 100 mM or lower. The higher the salt concentration used in the method of the invention, the shorter the one or more spacers that are typically used and vice versa.

Preferred combinations of features are shown in Table 4 below.

Spacer Spacer length Polynu- compo- (i.e. number Salt Free Helicase cleotide sition* of *) [ ] nucleotides? cofactor? DNA iSpC3 4 1M Yes Yes DNA iSp18 4 100- Yes Yes 1000 mM DNA iSp18 6 <100- Yes Yes 1000 mM DNA iSp18 2 1M Yes Yes DNA iSpC3 12 <100- Yes Yes 1000 mM DNA iSpC3 20 <100- Yes Yes 1000 mM DNA iSp9 6 100- Yes Yes 1000 mM DNA idSp 4 1M Yes Yes

The method may concern moving two or more helicases past a spacer. In such instances, the length of the spacer is typically increased to prevent the trailing helicase from pushing the leading helicase past the spacer in the absence of the pore and applied potential. If the method concerns moving two or more helicases past one or more spacers, the spacer lengths discussed above may be increased at least 1.5 fold, such 2 fold, 2.5 fold or 3 fold. For instance, if the method concerns moving two or more helicases past one or more spacers, the spacer lengths in the third column of Table 4 above may be increased 1.5 fold, 2 fold, 2.5 fold or 3 fold.

The two or more helicases may also be separated such that each has its own one or more spacers. This is discussed in more detail below.

Double Stranded Polynucleotide

The target polynucleotide may be double stranded. If the polynucleotide is double stranded, the invention preferably comprises attaching a bridging moiety adaptor, such as a hairpin loop adaptor, at one end of the polynucleotide and separating the two strands of the polynucleotide to form a single stranded polynucleotide construct. The single stranded polynucleotide construct may then be moved with respect to the pore, such as through the pore, in accordance with the invention. Linking and interrogating both strands on a double stranded construct in this way increases the efficiency and accuracy of characterisation.

The bridging moiety is capable of linking the two strands of the polynucleotide. The bridging moiety typically covalently links the two strands of the polynucleotide. The bridging moiety can be anything that is capable of linking the two strands of the polynucleotide, provided that the bridging moiety does not interfere with movement of the polynucleotide through the transmembrane pore.

The bridging moiety may be linked to the polynucleotide by any suitable means known in the art. The bridging moiety may be synthesized separately and chemically attached or enzymatically ligated to the polynucleotide. Alternatively, the bridging moiety may be generated in the processing of the polynucleotide.

The bridging moiety is linked to the polynucleotide at or near one end of the polynucleotide. The bridging moiety is preferably linked to the polynucleotide within 10 nucleotides of the end of the polynucleotide.

Although bridging moieties are preferred loading moieties, the bridging moiety does not have to have one or more bound (or attached) polynucleotide binding proteins bound as long as a loading moiety, such as a Y adaptor, has one or more bound (or attached) polynucleotide binding proteins attached to the other end of the target polynucleotide. In some embodiments, loading moieties may be attached at both ends of the polynucleotide of the invention, preferably where one is a Y adaptor and the other is a bridging moiety, such as a hairpin loop adaptor. Suitable bridging moieties are discussed above with reference to the loading moieties. If the hairpin loop is not being used as a loading moiety, the hairpin loop is typically 110 or fewer nucleotides, such as 100 or fewer nucleotides, 90 or fewer nucleotides, 80 or fewer nucleotides, 70 or fewer nucleotides, 60 or fewer nucleotides, 50 or fewer nucleotides, 40 or fewer nucleotides, 30 or fewer nucleotides, 20 or fewer nucleotides or 10 or fewer nucleotides, in length. The hairpin loop is preferably from about 1 to 110, from 2 to 100, from 5 to 80 or from 6 to 50 nucleotides in length. Longer lengths of the hairpin loop, such as from 50 to 110 nucleotides, are preferred if the loop is involved in the differential selectable binding of the adaptor. Similarly, shorter lengths of the hairpin loop, such as from 1 to 5 nucleotides, are preferred if the loop is not involved in the selectable binding as discussed below.

The bridging moiety adaptor or hairpin loop adaptor may be attached or ligated to the target polynucleotide as discussed above.

The two strands of the polynucleotide may be separated using any method known in the art. For instance, they may be separated by one or more polynucleotide binding proteins or using conditions which favour dehybridsation (examples of conditions which favour dehybridisation include, but are not limited to, high temperature, high pH and the addition of agents that can disrupt hydrogen bonding or base pairing, such as formamide and urea). If one or more polynucleotide binding proteins are used to separate the strands, the one or more polynucleotide binding proteins are typically attached to the target polynucleotide at the other end from the bridging moiety, for instance using a Y adaptor.

The one or more loading moieties, preferably the Y adaptor and/or the bridging moiety adaptor(such as the hairpin loop adaptor), preferably comprises a selectable binding moiety. This allows the polynucleotide to be purified or isolated. A selectable binding moiety is a moiety that can be selected on the basis of its binding properties. Hence, a selectable binding moiety is preferably a moiety that specifically binds to a surface. A selectable binding moiety specifically binds to a surface if it binds to the surface to a much greater degree than any other moiety used in the invention. In preferred embodiments, the moiety binds to a surface to which no other moiety used in the invention binds.

Suitable selective binding moieties are known in the art. Preferred selective binding moieties include, but are not limited to, biotin, a polynucleotide sequence, antibodies, antibody fragments, such as Fab and ScSv, antigens, polynucleotide binding proteins, poly histidine tails and GST tags. The most preferred selective binding moieties are biotin and a selectable polynucleotide sequence. Biotin specifically binds to a surface coated with avidins. Selectable polynucleotide sequences specifically bind (i.e. hybridise) to a surface coated with homologus sequences. Alternatively, selectable polynucleotide sequences specifically bind to a surface coated with polynucleotide binding proteins.

The one or more loading moieties, preferably the Y adaptor and/or the bridging moiety adaptor (such as the hairpin loop adaptor) and/or the selectable binding moiety may comprise a region that can be cut, nicked, cleaved or hydrolysed. Such a region can be designed to allow the first and/or second polynucleotide to be removed from the surface to which it is bound following purification or isolation. Suitable regions are known in the art. Suitable regions include, but are not limited to, an RNA region, a region comprising desthiobiotin and streptavidin, a disulphide bond and a photocleavable region.

Leader Sequence

The one or more loading moieties may be provided with a leader sequence which preferentially threads into the pore. The leader sequence facilitates the method of the invention. The leader sequence is designed to preferentially thread into the transmembrane pore and thereby facilitate the movement of target polynucleotide with respect to the pore, such as through the pore. The leader sequence can also be used to link the polynucleotide to the one or more anchors as discussed above.

The leader sequence typically comprises a polymer. The polymer is preferably negatively charged. The polymer is preferably a polynucleotide, such as DNA or RNA, a modified polynucleotide (such as abasic DNA), PNA, LNA, BNA, polyethylene glycol (PEG) or a polypeptide. The leader preferably comprises a polynucleotide and more preferably comprises a single stranded polynucleotide. The leader sequence can comprise any of the polynucleotides discussed above. The single stranded leader sequence most preferably comprises a single strand of DNA, such as a poly dT section. The leader sequence preferably comprises the one or more spacers.

The leader sequence can be any length, but is typically 10 to 150 nucleotides in length, such as from 20 to 150 nucleotides in length. The length of the leader typically depends on the transmembrane pore used in the method.

Double Coupling

In a preferred embodiment, the invention comprises a method for attaching one or more polynucleotide binding proteins to a target double stranded polynucleotide, comprising:

(a) providing a Y adaptor with one or more polynucleotide binding proteins bound (or attached) to it and one or more first anchors for coupling the polynucleotide to a membrane and providing a bridging moiety adaptor, such as a hairpin loop adaptor, with one or more second anchors wherein the strength of coupling of the bridging moiety adaptor to the membrane is greater than the strength of coupling of the Y adaptor to the membrane; and

(b) attaching the Y adaptor to one end of the target polynucleotide and attaching the bridging moiety to the other end of the target polynucleotide. The bridging moiety preferably has one or more polynucleotide binding proteins, preferably one or more molecular brakes, attached to it.

The invention also provides a method of characterising a target double stranded polynucleotide comprising

(c) contacting the polynucleotide provided in step (b) above with a transmembrane pore such that the one or more polynucleotide binding proteins control the movement of the target polynucleotide with respect to the pore, such as through the pore; and

(d) taking one or more measurements as the polynucleotide moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the polynucleotide and thereby characterising the target polynucleotide.

This type of method is discussed in detail in UK Application No. 1406147.7.

The Y adaptor and/or the bridging moiety adaptor are typically polynucleotide adaptors. They may be formed from any of the polynucleotides discussed above.

The Y adaptor typically comprises (a) a double stranded region and (b) a single stranded region or a region that is not complementary at the other end. The Y adaptor may be described as having an overhang if it comprises a single stranded region. The presence of a non-complementary region in the Y adaptor gives the adaptor its Y shape since the two strands typically do not hybridise to each other unlike the double stranded portion. The Y adaptor comprises the one or more first anchors. Anchors are discussed in more detail above.

The Y adaptor preferably comprises a leader sequence which preferentially threads into the pore. This is discussed above.

The bridging moiety adaptor preferably comprises a selectable binding moiety as discussed above. The bridging moiety adaptor and/or the selectable binding moiety may comprise a region that can be cut, nicked, cleaved or hydrolysed as discussed above.

The Y adaptor and/or the bridging moiety adaptor may be ligated to the polynucleotide using any method known in the art. One or both of the adaptors may be ligated using a ligase, such as T4 DNA ligase, E. coli DNA ligase, Taq DNA ligase, Tma DNA ligase and 9° N DNA ligase.

The strength of coupling (or binding) of the bridging moiety adaptor to the membrane is greater than the strength of coupling (or binding) of the Y adaptor to the membrane. This can be measured in any way. A suitable method for measuring the strength of coupling (or binding) is disclosed in the Examples of UK Application No. 1406147.7.

The strength of coupling (or binding) of the bridging moiety adaptor is preferably at least 1.5 times the strength of coupling (or binding) of the Y adaptor, such as at least twice, at least three times, at least four times, at least five or at least ten times the strength of coupling (or binding) of the Y adaptor. The affinity constant (Kd) of the bridging moiety adaptor for the membrane is preferably at least 1.5 times the affinity constant of the Y adaptor, such as at least twice, at least three times, at least four times, at least five or at least ten times the strength of coupling of the Y adaptor.

There are several ways in which the bridging moiety adaptor couples (or binds) more strongly to the membrane than the Y adaptor. For instance, the bridging moiety adaptor may comprise more anchors that than the Y adaptor. For instance, the bridging moiety adaptor may comprise 2, 3 or more second anchors whereas the Y adaptor may comprise one first anchor.

The strength of coupling (or binding) of the one or more second anchors to the membrane may be greater than the strength of coupling (or binding) of the one or more first anchors to the membrane. The strength of coupling (or binding) of the one or more second anchors to the bridging moiety adaptor may be greater than the strength of coupling (or binding) of the one or more first anchors to the Y adaptor. The one or more first anchors and the one or more second anchors may be attached to their respective adaptors via hybridisation and the strength of hybridisation is greater in the one or more second anchors than in the one or more first anchors. Any combination of these embodiments may also be used in the invention. Strength of coupling (or binding) may be measured using known techniques in the art.

The one or more second anchors preferably comprise one or more groups which couples(s) (or bind(s)) to the membrane with a greater strength than the one or more groups in the one or more first anchors which couple(s) (or bind(s)) to the membrane. In preferred embodiments, the bridging moiety adaptor/one or more second anchors couple (or bind) to the membrane using cholesterol and the Y adaptor/one or more first anchors couple (or bind) to the membrane using palmitate. Cholesterol binds to triblock copolymer membranes and lipid membranes more strongly than palmitate. In an alternative embodiment, the bridging moiety adaptor/one or more second anchors couple (or bind) to the membrane using a mono-acyl species, such as palmitate, and the Y adaptor/one or more first anchors couple (or bind) to the membrane using a diacyl species, such as dipalmitoylphosphatidylcholine.

Adding Hairpin Loops and Leader Sequences

In accordance with the invention, a double stranded polynucleotide may be contacted with a MuA transposase and a population of double stranded MuA substrates, wherein a proportion of the substrates in the population are Y adaptors bound to one or more polynucleotide binding proteins and comprising the leader sequence and wherein a proportion of the substrates in the population are bridging moiety adaptors, such as hairpin loop adaptors bound to one or more polynucleotide binding proteins. The Y adaptors and/or the bridging moiety adaptors function as the loading moieties. The transposase fragments the double stranded polynucleotide and ligates MuA substrates to one or both ends of the fragments. This produces a plurality of modified double stranded polynucleotides comprising the Y adaptor with the one or more polynucleotide binding proteins and leader sequence at one end and the bridging moiety (or hairpin loop) with the one or more polynucleotide binding proteins at the other. The modified double stranded polynucleotides may then be characterised using the method of the invention.

Each substrate in the population preferably comprises at least one overhang of universal nucleotides such that the transposase fragments the template polynucleotide and ligates a substrate to one or both ends of the double stranded fragments and thereby produces a plurality of fragment/substrate constructs and wherein the method further comprises ligating the overhangs to the fragments in the constructs and thereby producing a plurality of modified double stranded polynucleotides. Suitable universal nucleotides are discussed above. The overhang is preferably five nucleotides in length.

Alternatively, each substrate in the population preferably comprises (i) at least one overhang and (ii) at least one nucleotide in the same strand as the at least one overhang which comprises a nucleoside that is not present in the template polynucleotide such that the transposase fragments the template polynucleotide and ligates a substrate to one or both ends of the double stranded fragments and thereby produces a plurality of fragment/substrate constructs, and wherein the method further comprises (a) removing the overhangs from the constructs by selectively removing the at least one nucleotide and thereby producing a plurality of double stranded constructs comprising single stranded gaps and (b) repairing the single stranded gaps in the constructs and thereby producing a plurality of modified double stranded polynucleotides. The polynucleotide analyte typically comprises the nucleosides deoxyadenosine (dA), deoxyuridine (dU) and/or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC). The nucleoside that is not present in the polynucleotide is preferably abasic, adenosine (A), uridine (U), 5-methyluridine (m⁵U), cytidine (C) or guanosine (G) or comprises urea, 5, 6 dihydroxythymine, thymine glycol, 5-hydroxy-5 methylhydanton, uracil glycol, 6-hydroxy-5, 6-dihdrothimine, methyltartronylurea, 7, 8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, fapy-guanine, methy-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine, 5-hydroxy-uracil, 3-methyladenine, 7-methylguanine, 1,N6-ethenoadenine, hypoxanthine, 5-hydroxyuracil, 5-hydroxymethyluracil, 5-formyluracil or a cis-syn-cyclobutane pyrimidine dimer. The at least one nucleotide preferably is 10 nucleotides or fewer from the overhang. The at least one nucleotide preferably is the first nucleotide in the overhang. All of the nucleotides in the overhang preferably comprise a nucleoside that is not present in the template polynucleotide.

These MuA based methods are disclosed in UK Application No. 1314695.6. They are also discussed in detail in UK Application No. 1406147.7.

One or more helicases may be attached to the MuA substrate Y adaptors (i.e. loading moieties) before they are contacted with the double stranded polynucleotide and MuA transposase. Alternatively, one or more helicases may be attached to the MuA substrate Y adaptors (i.e. loading moieties) after they are contacted with the double stranded polynucleotide and MuA transposase.

One or more molecular brakes may be attached to the MuA substrate bridging moiety (or hairpin loop) adaptors before they are contacted with the double stranded polynucleotide and MuA transposase. Alternatively, one or more molecular brakes may be attached to the MuA substrate bridging moiety (or hairpin loop) adaptors after they are contacted with the double stranded polynucleotide and MuA transposase.

Polynucleotide Characterisation

The invention provides a method of characterising a target polynucleotide. The target polynucleotide may also be called the template polynucleotide or the polynucleotide of interest. The method of the invention involves measuring one or more characteristics of the polynucleotide. In particular, one of the methods above for controlling the movement of a polynucleotide through a transmembrane pore is carried out as step (a) and then in step (b) one or more measurements are taken as the polynucleotide moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the polynucleotide. Suitable measurements are discussed above.

Any number of target polynucleotides can be investigated. For instance, the method of the invention may concern characterising 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more target polynucleotides. The target polynucleotide can be naturally occurring or artificial. For instance, the method may be used to verify the sequence of manufactured oligonucleotides. The methods are typically carried out in vitro.

The method may involve measuring one, two, three, four or five or more characteristics of the target polynucleotide. The one or more characteristics are preferably selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide and (v) whether or not the polynucleotide is modified. Any combination of (i) to (v) may be measured in accordance with the invention, such as {i}, {ii}, {iii}, {iv}, {v}, {i,ii}, {i,iii}, {i,iv}, {i,v}, {ii,iii}, {ii,iv}, {ii,v}, {iii,iv}, {iii,v}, {iv,v}, {i,ii,iii}, {i,ii,iv}, {i,ii,v}, {i,iii,iv}, {i,iii,v}, {i,iv,v}, {ii,iii,iv}, {ii,iii,v}, {ii,iv,v}, {iii,iv,v}, {i,ii,iii,iv}, {i,ii,iii,v}, {i,ii,iv,v}, {i,iii,iv,v}, {ii,iii,iv,v} or {i,ii,iii,iv,v}.

For (i), the length of the polynucleotide may be measured for example by determining the number of interactions between the polynucleotide and the pore or the duration of interaction between the polynucleotide and the pore.

For (ii), the identity of the polynucleotide may be measured in a number of ways. The identity of the polynucleotide may be measured in conjunction with measurement of the sequence of the polynucleotide or without measurement of the sequence of the polynucleotide. The former is straightforward; the polynucleotide is sequenced and thereby identified. The latter may be done in several ways. For instance, the presence of a particular motif in the polynucleotide may be measured (without measuring the remaining sequence of the polynucleotide). Alternatively, the measurement of a particular electrical and/or optical signal in the method may identify the polynucleotide as coming from a particular source.

For (iii), the sequence of the polynucleotide can be determined as described previously. Suitable sequencing methods, particularly those using electrical measurements, are described in Stoddart D et al., Proc Natl Acad Sci, 12;106(19):7702-7, Lieberman K R et al, J Am Chem Soc. 2010; 132(50):17961-72, and International Application WO 2000/28312.

For (iv), the secondary structure may be measured in a variety of ways. For instance, if the method involves an electrical measurement, the secondary structure may be measured using a change in dwell time or a change in current flowing with respect to the pore, such as through the pore. This allows regions of single-stranded and double-stranded polynucleotide to be distinguished.

For (v), the presence or absence of any modification may be measured. The method preferably comprises determining whether or not the polynucleotide is modified by methylation, by oxidation, by damage, with one or more proteins or with one or more labels, tags or spacers. Specific modifications will result in specific interactions with the pore which can be measured using the methods described below. For instance, methylcyotsine may be distinguished from cytosine on the basis of the current flowing with respect to the pore, such as through the pore, during its interaction with each nucleotide.

The methods may be carried out using any apparatus that is suitable for investigating a membrane/pore system in which a pore is present in a membrane. The method may be carried out using any apparatus that is suitable for transmembrane pore sensing. For example, the apparatus comprises a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections. The barrier typically has an aperture in which the membrane containing the pore is formed. Alternatively the barrier forms the membrane in which the pore is present.

The methods may be carried out using the apparatus described in International Application No. PCT/GB08/000562 (WO 2008/102120).

The methods may involve measuring the current passing through the pore as the polynucleotide moves with respect to the pore. Therefore the apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore. The methods may be carried out using a patch clamp or a voltage clamp. The methods preferably involve the use of a voltage clamp.

The methods of the invention may involve the measuring of a current passing through the pore as the polynucleotide moves with respect to the pore. Suitable conditions for measuring ionic currents through transmembrane protein pores are known in the art and disclosed in the Example. The method is typically carried out with a voltage applied across the membrane and pore. The voltage used is typically from +5 V to −5 V, such as from +4 V to −4 V, +3 V to −3 V or +2 V to −2 V. The voltage used is typically from −600 mV to +600 mV or −400 mV to +400 mV. The voltage used is preferably in a range having a lower limit selected from −400 mV, −300 mV, −200 mV, −150 mV, −100 mV, −50 mV, −20 mV and 0 mV and an upper limit independently selected from +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used is more preferably in the range 100 mV to 240 mV and most preferably in the range of 120 mV to 220 mV. It is possible to increase discrimination between different nucleotides by a pore by using an increased applied potential.

The methods are typically carried out in the presence of any charge carriers, such as metal salts, for example alkali metal salt, halide salts, for example chloride salts, such as alkali metal chloride salt. Charge carriers may include ionic liquids or organic salts, for example tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazolium chloride. In the exemplary apparatus discussed above, the salt is present in the aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl), caesium chloride (CsCl) or a mixture of potassium ferrocyanide and potassium ferricyanide is typically used. KCl, NaCl and a mixture of potassium ferrocyanide and potassium ferricyanide are preferred. The charge carriers may be asymmetric across the membrane. For instance, the type and/or concentration of the charge carriers may be different on each side of the membrane.

The salt concentration may be at saturation. The salt concentration may be 3 M or lower and is typically from 0.1 to 2.5 M, from 0.3 to 1.9 M, from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to 1.6 M or from 1 M to 1.4 M. The salt concentration is preferably from 150 mM to 1 M. The method is preferably carried out using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. High salt concentrations provide a high signal to noise ratio and allow for currents indicative of the presence of a nucleotide to be identified against the background of normal current fluctuations.

The methods are typically carried out in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in the aqueous solution in the chamber. Any buffer may be used in the method of the invention. Typically, the buffer is phosphate buffer. Other suitable buffers are HEPES and Tris-HCl buffer. The methods are typically carried out at a pH of from 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from 5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pH used is preferably about 7.5.

The methods may be carried out at from 0° C. to 100° C., from 15° C. to 95° C., from 16° C. to 90° C., from 17° C. to 85° C., from 18° C. to 80° C., 19° C. to 70° C., or from 20° C. to 60° C. The methods are typically carried out at room temperature. The methods are optionally carried out at a temperature that supports enzyme function, such as about 37° C.

Bridging Moiety Sequencing

In a preferred embodiment, a target double stranded polynucleotide is provided with a bridging moiety (or hairpin loop) adaptor at one end and the method comprises contacting the polynucleotide with a transmembrane pore such that both strands of the polynucleotide move with respect to the pore, such as through the pore, and taking one or more measurements as the both strands of the polynucleotide move with respect to the pore wherein the measurements are indicative of one or more characteristics of the strands of the polynucleotide and thereby characterising the target double stranded polynucleotide. Any of the embodiments discussed above equally apply to this embodiment.

Modified Polynucleotides

Before use in the method of the invention, a polynucleotide may be modified by contacting the polynucleotide with a polymerase and a population of free nucleotides under conditions in which the polymerase forms a modified polynucleotide using the polynucleotide as a template, wherein the polymerase replaces one or more of the nucleotide species in the polynucleotide with a different nucleotide species when forming the modified polynucleotide. The modified polynucleotide may then be used in the methods of the invention. This type of modification is described in UK Application No. 1403096.9. Any of the polymerases discussed above may be used. The polymerase is preferably Klenow or 9° North.

The polynucleotide is contacted with the polymerase under conditions in which the polymerase forms a modified polynucleotide using the polynucleotide as a template. Such conditions are known in the art. For instance, the polynucleotide is typically contacted with the polymerase in commercially available polymerase buffer, such as buffer from New England Biolabs®. The temperature is preferably from 20 to 37° C. for Klenow or from 60 to 75° C. for 9° North. A primer or a 3′ hairpin is typically used as the nucleation point for polymerase extension. The polymerase may be contacted with the target polynucleotide in accordance with the invention, i.e. by (a) providing the one or more polymerases bound to one or more loading moieties and (b) attaching the one or more loading moieties to the target polynucleotide.

Characterisation, such as sequencing, of a polynucleotide using a transmembrane pore typically involves analyzing polymer units made up of k nucleotides where k is a positive integer (i.e. ‘k-mers’). This is discussed in International Application No. PCT/GB2012/052343 (published as WO 2013/041878). While it is desirable to have clear separation between current measurements for different k-mers, it is common for some of these measurements to overlap. Especially with high numbers of polymer units in the k-mer, i.e. high values of k, it can become difficult to resolve the measurements produced by different k-mers, to the detriment of deriving information about the polynucleotide, for example an estimate of the underlying sequence of the polynucleotide.

By replacing one or more nucleotide species in the polynucleotide with different nucleotide species in the modified polynucleotide, the modified polynucleotide contains k-mers which differ from those in the polynucleotide. The different k-mers in the modified polynucleotide are capable of producing different current measurements from the k-mers in the polynucleotide and so the modified polynucleotide provides different information from the polynucleotide. The additional information from the modified polynucleotide can make it easier to characterise the polynucleotide. In some instances, the modified polynucleotide itself may be easier to characterise. For instance, the modified polynucleotide may be designed to include k-mers with an increased separation or a clear separation between their current measurements or k-mers which have a decreased noise.

The polymerase preferably replaces two or more of the nucleotide species in the polynucleotide with different nucleotide species when forming the modified polynucleotide. The polymerase may replace each of the two or more nucleotide species in the polynucleotide with a distinct nucleotide species. The polymerase may replace each of the two or more nucleotide species in the polynucleotide with the same nucleotide species.

If the polynucleotide is DNA, the different nucleotide species in the modified polynucleotide typically comprises a nucleobase which differs from adenine, guanine, thymine, cytosine or methylcytosine and/or comprises a nucleoside which differs from deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine or deoxymethylcytidine. If the polynucleotide is RNA, the different nucleotide species in the modified polynucleotide typically comprises a nucleobase which differs from adenine, guanine, uracil, cytosine or methylcytosine and/or comprises a nucleoside which differs from adenosine, guanosine, uridine, cytidine or methylcytidine. The different nucleotide species may be any of the universal nucleotides discussed above.

The polymerase may replace the one or more nucleotide species with a different nucleotide species which comprises a chemical group or atom absent from the one or more nucleotide species. The chemical group may be a propynyl group, a thio group, an oxo group, a methyl group, a hydroxymethyl group, a formyl group, a carboxy group, a carbonyl group, a benzyl group, a propargyl group or a propargylamine group.

The polymerase may replace the one or more nucleotide species with a different nucleotide species which lacks a chemical group or atom present in the one or more nucleotide species. The polymerase may replace the one or more of the nucleotide species with a different nucleotide species having an altered electronegativity. The different nucleotide species having an altered electronegativity preferably comprises a halogen atom.

The method preferably further comprises selectively removing the nucleobases from the one or more different nucleotides species in the modified polynucleotide.

Products

The invention also provides a target polynucleotide modified using the invention. The invention also provides a loading moiety having one or more polynucleotide binding proteins bound to it. Any of the embodiments discussed above with reference to the method of the invention apply to the polynucleotide and moiety of the invention.

Kits

The present invention also provides a kit for attaching one or more polynucleotide binding proteins to a target polynucleotide, comprising (a) the one or more polynucleotide binding proteins bound to one or more loading moieties and (b) a ligase. Any of the embodiments discussed above apply to the kits.

The kit is preferably for a double stranded polynucleotide through a transmembrane pore and the kit preferably comprises a Y adaptor having one or more helicases attached and a bridging moiety (or hairpin loop) adaptor having one or more molecular brakes attached. The Y adaptor preferably comprises one or more first anchors for coupling the polynucleotide to the membrane, the bridging moiety (or hairpin loop) adaptor preferably comprises one or more second anchors for coupling the polynucleotide to the membrane and the strength of coupling of the bridging moiety (or hairpin loop) adaptor to the membrane is preferably greater than the strength of coupling of the Y adaptor to the membrane.

The kit preferably further comprises a transmembrane pore. Any of the membrane and pores discussed above may be in the kit.

Any of the embodiments discussed above with reference to the method of the invention equally apply to the kits. The kit may further comprise the components of a membrane, such as the components of an amphiphilic layer or a triblock copolymer membrane.

The kit of the invention may additionally comprise one or more other reagents or instruments which enable any of the embodiments mentioned above to be carried out. Such reagents or instruments include one or more of the following: suitable buffer(s) (aqueous solutions), means to obtain a sample from a subject (such as a vessel or an instrument comprising a needle), means to amplify and/or express polynucleotides, a membrane as defined above or voltage or patch clamp apparatus. Reagents may be present in the kit in a dry state such that a fluid sample resuspends the reagents. The kit may also, optionally, comprise instructions to enable the kit to be used in the method of the invention or details regarding for which organism the method may be used.

The following Examples illustrate the invention.

Example 1

This example describes the sample preparation procedure for binding a polynucleotide binding protein to a loading moiety.

Materials and Methods

TrwC Cba-L376C/Q594A/K762C (6.5 μM, SEQ ID NO: 25 with mutations L376C/Q594A/K762C) and DNA hairpin-adapter (4 μM, SEQ ID NO: 29 was attached at the 3′ end to four iSpC3 spacers which are attached at the opposite end to the 5′ end of SEQ ID NO: 30) were mixed in buffer (100 mM KCl, 100 mM CAPS (pH 10) and 1 mM EDTA) and incubated for 30 minutes. TRIS (0.1 volumes of 1 M TRIS pH 7.0) was added to the DNA/enzyme mixture and the mixture thoroughly mixed. Finally, 0.025 volumes of 1.29 mM Bismaleimideoethane in DMF was added and the mixture incubated for a further 15 minutes. The final concentrations of each component were as follows TrwC Cba-L376C/Q594A/K762C (5.8 μM), DNA Hairpin-adapter (3.56 μM), Buffer (89 mM KCl, 89 mM CAPS, pH 10, 0.89 mM EDTA, 89 mM Tris (pH 7)) and Bismaleimideoethane (28.7 μM), this mixture was known as sample 1.

The pre-bound TrwC Cba-L376C/Q594A/K762C to the DNA Hairpin-adapter was then purified using the procedure described using an 8 mL POROS HQ-10 column (FPLC) and the following elution buffers (Buffer A—50 mM Ethanolamine, 300 mM NaCl, 0.1% 13-OTG, 1 mM TCEP, pH 10.0, Buffer B—50 mM Ethanolamine, 700 mM NaCl, 0.1% 13-OTG, 1 mM TCEP, pH 10.0). Sample 1 was loaded onto the column and any DNA which did not have TrwC Cba-L376C/Q594A/K762C bound or enzyme which was not bound to DNA was washed off the column using 5 column volumes of buffer A. The pre-bound TrwC Cba-L376C/Q594A/K762C to the DNA Hairpin-adapter was then eluted with 0−100% buffer B in 11.8 column volumes, this sample was known as Sample 2. An example FPLC trace is shown in FIG. 1 the peak labelled P1 corresponded to pre-bound TrwC Cba-L376C/Q594A/K762C to the DNA Hairpin-adapter.

Sample 2 was then further purified using a 5 mL Histrap HP column (FPLC) using the following elution buffers (Buffer C—20 mM Na-CAPS, 100 mM NaCl, 0.1% 13-OTG, 1 mM TCEP, pH 10.0, Buffer D—20 mM Na-CAPS, 2M NaCl, 10% (w/v) glycerol, 0.1% 13-OTG, 1 mM TCEP, pH 10.0 and Buffer E—20 mM Na-CAPS, 100 mM NaCl, 300 mM imidazole, 0.1% 13-OTG, 1 mM TCEP, pH 10.0). Sample 2 was loaded onto the column and any DNA which did not have TrwC Cba-L376C/Q594A/K762C bound or enzyme which was not bound to DNA was washed off the column with buffer C. The column was then washed with Buffer D and then washed again with Buffer C. Finally the pre-bound TrwC Cba-L376C/Q594A/K762C to the DNA Hairpin-adapter was then eluted with 0−100% buffer E in ten column volumes. The main elution peak (an example is shown in FIG. 2 with the main peak labelled P2) was then pooled, the concentration measured and the DNA sample analysed using a TBE (Native) PAGE and an SDS PAGE gel. The TBE (native) PAGE was 4−20% TBE gel, run at 150 V for 25 minutes and then stained using SYBR gold stain. This stain allowed the visibility of any DNA in the sample (with or without enzyme bound). FIG. 3 shows this gel. Columns 4−6 show that after purification the DNA was still bound to the enzyme. The SDS PAGE was 10% Bis-Tris gel, XT MOPS, run at 200 V run for 60 min and then stained using SYPRO ruby stain. This stain allowed the visibility of any enzyme in the sample (bound or not bound to DNA). FIG. 4 shows in columns 4−6 that after purification the enzyme was closed onto the DNA using the Bismaleimideoethane linker.

Example 2

This example describes the sample preparation procedure for binding a polynucleotide binding protein to a loading moiety.

Materials and Methods

T4 Dda-E94C/C109A/C136A/A360C (3 μM, SEQ ID NO: 24 with mutations E94C/C109A/C136A/A360C) and DNA Y-adapter (500 nM, SEQ ID NO: 26 was hybridised to DNA strand X=30 iSpC3 spacers attached at the 3′ end to the 5′ end SEQ ID NO: 27 which is attached at the 3′ end to 4 iSp18 spacers which are attached at the opposite end to the 5′ end of SEQ ID NO: 28) were mixed in buffer (50 mM HEPES, 100 mM KOAc (pH 8) and EDTA (1 mM) and TMAD (100 μM) were added and incubated for 60 minutes at room temperature. The mixture was then diluted 1:1 in KCl/ATP solution (500 mM KCl, 1 mM ATP final) and incubated at ambient temperature for 25 mins, this mixture was known as sample 3.

This sample was then purified using SPRI beads. The SPRI beads were prepared using the procedure below—

-   -   1. A serological pipette was used to transfer Wash Buffer (30         mL, 50 mM Tris pH7.5, 2.5 M NaCl) into a 50 mL Falcon tube.     -   2. The stock pot of Sera-Mag Speed Bead Carboxylate-Modified         Magnetic Particles (Thermo) was vortexed to resuspend the beads.     -   3. The bead suspension (600 μL) was transferred into the 30 mL         of Wash Buffer (Step 1) and vortexed to resuspend the beads.     -   4. The Falcon tube was then placed into DynaMag-50 magnetic         rack, the beads were allowed to accumulate at the side of the         tube adjacent to the magnet, and the supernatant was removed         using a serological pipette.     -   5. A further 30 mL of Wash Buffer was added to the tube and the         tube was vortexed thoroughly to resuspend the beads.     -   6. The Falcon tube was then placed back into the DynaMag-50         magnetic rack and the beads were allowed to accumulate at the         side of the tube, adjacent to the magnet.     -   7. The supernatant was removed from the tube once the solution         had cleared.     -   8. Steps 5, 6 and 7 were repeated in sequence three more times.     -   9. The Binding Buffer (30 mL, 50 mM Tris pH7.5, 2.5 M NaCl, 20%         PEG8000) was added to the Falcon tube from Step 8 and the tube         was then vortexed to resuspend the beads.

The beads were then stored at 4° C. prior to use, although they were used under ambient conditions during purification.

Sample 3 was purified using the procedure outlined below—

-   -   1. The tube from Step 9 above was then vortexed in order to         resuspend beads, and 18.5 mL (of sample 3) was transferred into         a clean 50 mL Falcon tube.     -   2. The tube was placed in DynaMag-50 magnetic rack and the beads         were allowed to accumulate on the side of the tube, adjacent to         the magnet. Note that since the beads were suspended in Binding         Buffer (50 mM Tris pH7.5, 2.5 M NaCl, 20% PEG8000) the         accumulation could have taken in excess of 10 minutes.     -   3. The supernatant was removed once the solution had cleared,         18.5 mL of Binding Buffer was added and the sample was vortexed         thoroughly to resuspend the beads.     -   4. The tube was placed in a DynaMag-50 magnetic rack and the         beads were allowed to accumulate on the side of the tube,         adjacent to the magnet. Note that since the beads were suspended         in Binding Buffer (50 mM Tris pH7.5, 2.5 M NaCl, 20% PEG8000)         the accumulation of beads could have taken in excess of 10         minutes.     -   5. Step 3 was then repeated.

The sample was not pipette-mixed or vortexed from this point onwards.

-   -   6. The Falcon tube was placed back into a DynaMag-50 magnetic         rack and the beads were allowed to accumulate on the side of the         tube adjacent to the magnet. Note that since the buffer         contained PEG8000, the accumulation of beads could have taken in         excess of 10 minutes.     -   7. The supernatant was removed using a serological pipette and         replaced with enough Binding Buffer to cover the beads (−25 mL).     -   8. The Falcon tube was left in the DynaMag-50 magnetic rack and         the tube was rotated clockwise on its axis through 90°, allowing         the accumulated beads to move around the sides of the tube until         they settled at a new location adjacent to the magnets.     -   9. Step 8 was repeated three more times until the Falcon tube         had returned to its original position.     -   10. The supernatant was removed using serological pipette, and         then as much residual supernatant as possible was removed.     -   11. The Falcon tube was removed from the DynaMag-50 magnetic         rack and Elution Buffer (2.5 mL, 20 mM NaCl, 50 mM Tris, pH7.5)         was added.     -   12. The tube was gently agitated by flicking to resuspend the         beads and then the sample was incubated under ambient conditions         for 5 min.     -   13. The Falcon tube was returned to the DynaMag-50 magnetic         rack, the beads were allowed to accumulate on the side of the         tube adjacent to the magnets and supernatant was transferred to         a 5 mL Protein LoBind microcentrifuge tube.     -   14. The eluted material from Step 13 was diluted two-fold         through the addition of Elution Buffer.     -   15. The material was stored at 4° C. before it was aliquoted         into tubes.

The resultant enzyme bound to DNA was then analysed using a TBE PAGE gel as described previously in Example 1 except the gel was run for 45 mins. The gel is shown in FIG. 5 and column 3 shows the SPRI purification purified a single enzyme bound to the DNA Y-adapter.

Example 3

This example describes the sample preparation procedure for ligation of the enzyme pre-bound to the Y-adapter and the enzyme bound to the hairpin adapter to a random strand of genomic double-stranded DNA.

Materials and Methods

The random genomic double-stranded DNA sequence was dA tailed using a NEBNext dA-tailing module (NEB). No further purification was necessary. The dA-tailed genomic DNA (30 μL) was mixed with Y-adapter DNA with T4 Dda-E94C/C109A/C136A/A360C pre-bound (10 μL 200 nM) and hairpin-adapter with TrwC Cba-L376C/Q594A/K762C pre-bound (10 μL, 1 μM) and Blunt/TA ligase master mix (50 μL) and the sample was inverted 10 times to mix. The sample was then briefly spun down in a microfuge. The sample was then incubated for 10 mins at room temperature. The adapted DNA was purified using 0.4× Agencourt AMPure XP beads by volume, following the manufacturer's protocol, but using the following Wash Buffer (750 mM NaCl, 10% PEG 8000, 50 mM Tris.HCl pH 8) and Elution Buffer (40 mM CAPS pH 10, 40 mM KCl along with appropriate DNA tethers) as detailed below.

After following the manufactures protocol the pelleted beads were bathed in 150 μl Wash Buffer for 30 sec. The Wash Buffer was then carefully aspirated taking care not to disturb the pellet. The sample was then briefly spun in a microfuge, in order to sufficiently drain excess Wash Buffer from the beads. The tube was then replaced on the magnet to pellet the beads and then left for approximately 1−2 min. Finally the remaining Wash Buffer was then aspirated and the pelleted beads resuspended in 25 μl of Elution Buffer and mixed thoroughly. The sample was then left for 10 min before pelleting and removing the eluate. Finally, a DNA strand with a cholesterol attached at one end was hybridised to the sample. This was the sample DNA which was then tested in Example 4.

Example 4

This example illustrated that after the above sample preparation procedure the DNA construct which was produced had functioning pre-bound enzymes (T4 Dda-E94C/C109A/C136A/A360C and TrwC Cba-L376C/Q594A/K762C) which were capable of controlling the movement of the DNA through an MspA nanopore.

Materials and Methods

The DNA construct produced in Example 3 (final concentration added to the nanopore system 0.1 nM) which had both enzymes pre-bound (see FIG. 6 data) was added to buffer (final concentrations added to the nanopore system were 500 mM KCl, 25 mM potassium phosphate pH 8.0), ATP (final concentration added to the nanopore system 2 mM) and MgCl2 (final concentration added to the nanopore system 2 mM). This was the pre-mix which was then added to the nanopore system (total volume 150 μL).

Electrical measurements were acquired from single MspA nanopores inserted in block co-polymer in buffer (25 mM potassium phosphate, 150 mM potassium ferrocyanide (II), 150 mM potassium ferricyanide (III), pH 8.0). After achieving a single pore inserted in the block co-polymer, then buffer (2 mL, 25 mM potassium phosphate, 150 mM potassium ferrocyanide (II), 150 mM potassium ferricyanide (III), pH 8.0) was flowed through the system to remove any excess MspA nanopores. The enzyme pre-bound to construct Y (with both enzymes T4 Dda-E94C/C109A/C136A/A360C and TrwC Cba-L376C/Q594A/K762C pre-bound), fuel (MgCl2 and ATP) pre-mix (150 μL total) was then flowed into the single nanopore experimental system and the experiment run at a holding potential of −120 mV for 6 hours (with potential flips to +60 mV for 2 seconds) and helicase-controlled DNA movement monitored.

Results

Helicase controlled DNA movement was observed for the DNA construct Y using both T4 Dda-E94C/C109A/C136A/A360C and TrwC Cba-L376C/Q594A/K762C in tandem (see FIG. 6 ). The Figure shows the controlled translocation of regions 1 and 2 labelled in the figure, which corresponded to the random genomic double stranded DNA. When the spacers which were present in the hairpin adapter translocated through the nanopore increased current flow was observed see label 3. Therefore this sample helicase controlled DNA movement showed that the sample preparation procedure was successful as regions 1 and 2 translocated through the nanopore under the control of the enzymes and the increased current flow spike was used to clearly identify the transition between the regions.

Example 5: MuA Pre-Loaded Enzyme Adapter

In this example we showed that Enzyme can be pre-bound to the MuA Adaptor and this does not affect the function of MuA i.e., MuA can still attach the adaptor to the DNA.

T4 Dda-E94C/C109A/C136A/A360C (SEQ ID NO: 24 with mutations 4C/C109A/C136A/A360C) was pre-bound to the MuA Y-adapter: As illustrated in FIG. 7 , the adapter peak shifts (left is non-prebound and right is prebound).

Tagmentation uses a transposase to fragment and attach adapters to genomic DNA. The conditions for tagmentation were as follows: The adapter was pre-loaded with Enzyme. Tagmentation was carried out using: 100 nM MuA Tetramer with 400 nM T4 Dda-E94C/C109A/C136A/A360C (SEQ ID NO: 24 with mutations 4C/C109A/C136A/A360C) prebound adapter. 25 ng ul-1 Target lambda DNA. 25 mM Tris-HCl pH 8.0 at 20° C., 10 mM MgCl2, 110 mM NaCl, 0.05% Triton X-100, 10% glycerol 30° C. for 1 hour.

To visualise on Agilent Bioanalyser, T4 Dda-E94C/C109A/C136A/A360C (SEQ ID NO: 24 with mutations 4C/C109A/C136A/A360C) and MuA were heat inactivated at 75° C. for 10 mins. As illustrated in FIG. 8 , no adverse effect was seen on the Tagmentation of the target DNA. This can be seen by the fact the target DNA smears (left is non-prebound and right is prebound). The results show that it does not matter if Enzyme was attached, the fragmented genomic DNA has the same distribution of fragment sizes.

The DNA sample produced using the tagmentation protocol described previously was tested using a similar procedure as described in Example 4 using only T4 Dda-E94C/C109A/C136A/A360C as the helicase. As shown in FIG. 9 , the resultant sample library can be added onto a chip directly and helicase controlled DNA movement was observed.

Example 6

Example describes loading of two different enzymes onto two DNA components, which were then ligated together and attached to genomic DNA. The example below describes the loading of two different enzymes, however, this procedure is equally applicable for attaching two enzymes which are the same.

Materials and Methods

The same protocol as described in Example 2 was followed and two enzymes (E1=T4 Dda-(H82R/E94C/A360C) (SEQ ID NO: 24 with mutations H82R/E94C/A360C) and E2=T4 Dda-E94C/C109A/C136A/A360C (SEQ ID NO: 24 with mutations E94C/C109A/C136A/A360C) in FIG. 10 ) were separately loaded onto two DNA constructs (A-piece and END-piece in FIG. 10 ), and subsequently purified as described in Example 2. The A-piece comprised two DNA oligos hybridized together (see the figure legend of FIG. 10 for a full description of this oligonucleotide). The END-piece comprised three DNA oligos hybridized together (see the figure legend of FIG. 10 for a full description of this oligonucleotide).

The purified enzyme-loaded A-piece and END-piece DNA adapters were then ligated to a 3.6 kb section of genomic lambda dsDNA (template and complement SEQID), along with the hairpin adapter 3 (SEQ ID NO: 39 with a 5′ phosphate which was attached at the 3′ end to four iSpC3 spacers which are attached at the opposite end to the 5′ end of SEQ ID NO: 40) using the protocol described below.

The 3.6 kb genomic DNA (0.34 μL, 5 nM final, SEQ ID NO: 41 hybridised to SEQ ID NO: 42) was mixed with A-piece E1-DNA (6.25 uL, 50 nM final) END-piece E2-DNA (3.125 uL, 25 nM final), hairpin 3 (1.25 uL, 25 nM final) with ligation buffer (5 uL, 2 mM ATPγS, 4 mM MgCl2, 10 mM Hepes pH8.0, 6% PEG 8000, 10 mM NaCl final), Quick T4 DNA Ligase NEB (2.5 uL, 200 U/uL final) and nuclease free water (8 uL). The sample was incubated for 10 mins at room temperature. Finally, a DNA strand with a cholesterol attached at one end was hybridised to the sample. The sample 4 was not purified further and used in nanopore electrophysiology as described in Example 4.

Results

Helicase controlled DNA movement was observed for the DNA construct in sample 4 (see DNA construct shown in FIG. 11 ) using both T4 Dda-(H82R/E94C/A360C) and T4 Dda-E94C/C109A/C136A/A360C in tandem (see FIG. 11 ). The Figure shows the controlled translocation of regions 1 and 2 labelled in the figure, which corresponded to the genomic double stranded DNA. When the spacers which were present in the hairpin adapter translocated through the nanopore increased current flow was observed see arrow label 3. Therefore this sample helicase controlled DNA movement showed that the sample preparation procedure was successful as regions 1 and 2 translocated through the nanopore under the control of the enzymes and the increased current flow spike was used to clearly identify the transition between the regions.

Example 7

This example describes pre-loading a helicase and a polymerase onto a loading moiety, which was then ligated to a 3.6 kB strand of DNA. The polymerase was then used to make a copy of both the template and the complement strands of the 3.6 kB strand of DNA. A cartoon representation of the ligation and polymerisation steps is shown in FIG. 17 .

Materials and Methods

The helicase-leader complex (diagram shown in FIG. 12 section A) was prepared as described in Example 2, except SEQ ID NO: 26 is replaced with SEQ ID NO: 43. FIG. 15 lane 3 shows this sample run on a TBE PAGE showing the enzyme had bound to the DNA (band labelled c).

The polymerase-strand complex (diagram shown in FIG. 12 section B) was prepared by pre-binding Phi29-A411C/Q560C (SEQ ID NO: 9 with mutations A411C/Q560C, labelled X1) onto the DNA hairpin strand (SEQ ID NO: 44 attached at its 3′ end to four iSpC3 spacers which are attached at the opposite end to the 5′ end of SEQ ID NO: 45). Phi29-A411C/Q560C was buffer exchanged using Zeba 0.5 ml desalting columns (89882, Piercenet) in accordance with the protocol (https://www.piercenet.com/instructions/2161515.pdf) into the following buffer (50 mM tris pH 8, 20 mM (NH₄)₂SO₄, 10 mM MgCl₂, 4% glycerol) and diluted to 400 nM. The DNA hairpin strand (SEQ ID NO: 44 attached at its 3′ end to four iSpC3 spacers which are attached at the opposite end to the 5′ end of SEQ ID NO: 45) was added to the Phi29-A411C/Q560C which produced a sample (400 nM) with a 1:1 ratio of Phi29-A411C/Q560C:DNA hairpin strand (this sample corresponded to band E in FIG. 15 ). This was incubated for 15 minutes at room temperature. TMAD (125 uM) was then added and the sample incubated for a further 15 minutes at room temperature. The sample was then buffer exchanged again as described previously except the buffer used was (10 mM HEPES pH8, 10 mM MgCl₂). This sample was then mixed 1:1 with helicase-leader complex (the helicase-leader complex corresponded to band C in FIG. 15 ) to create the helicase/polymerase leader complex shown in section C of FIG. 12 .

The helicase/polymerase leader complex was ligated onto the 3.6 kb DNA strand (SEQ ID NO: 46) at 2:1 excess, with 2:1 excess of hairpin (SEQ ID NO: 47). Ligation was carried out in 5× ATP ligation buffer (5×: 150 mM Tris pH8, 50 mM MgCl₂, 5 mM ATP, 30% PEG 8000) in the presence of 10% T4 quick ligase. The sample was incubated for 15 mins at room temperature. The construct produced after the ligation step is shown in FIG. 13 . For the polymerase fill-in step, dNTPs (0.5 mM) were added. The sample was then incubated at 30° C. for 1 hour. Finally, SEQ ID NO: 31 was added at a 5×excess to the sample and the sample incubated from a minimum of 15 mins at room temperature.

The final sample 5 was not purified further and used in nanopore electrophysiology as described in Example 4.

Results

Helicase controlled DNA movement was observed for the DNA construct in sample 5 (see DNA construct shown in FIG. 13 ) using T4 Dda-E94C/C109A/C136A/A360C in (see FIG. 14 ). The Figure shows the controlled translocation of regions 1, 2, 4 and 5 labelled in the figure, which corresponded to the original 3.6 kB DNA strand (section 1 and 2) and the complementary strand produced by the polymerase (section 4 and 5). When the spacers which were present in the hairpin of the final construct (shown as x and labelled 3 in the top construct diagram of FIG. 14 ) translocated through the nanopore increased current flow was observed see arrow label 3. Therefore this sample helicase controlled DNA movement showed that the sample ligation and polymerisation preparation procedure was successful as regions 1, 2 and polymerised regions 4 and 5 translocated through the nanopore under the control of the enzyme and the increased current flow spike was used to clearly identify the transition between the original strand regions and the polymerised strand regions. 

The invention claimed is:
 1. A method for attaching one or more polynucleotide binding proteins to a target polynucleotide, comprising: (a) providing a complex formed by the one or more polynucleotide binding proteins and one or more loading moieties comprising a loading polynucleotide, wherein the one or more polynucleotide binding proteins are bound to the loading polynucleotide; and (b) attaching the loading polynucleotide of the one or more loading moieties of the complex to the target polynucleotide, wherein the one or more polynucleotide binding proteins of the complex retain their function and ability to bind to the loading polynucleotide of the one or more loading moieties after said attaching the loading polynucleotide of the one or more loading moieties of the complex to the target polynucleotide; and wherein the one or more polynucleotide binding proteins are one or more helicases, and wherein the one or more helicases are He1308 helicases, RecD helicases, XPD helicases or Dda helicases.
 2. A method for attaching one or more polynucleotide binding proteins to a target polynucleotide, comprising: (a) providing a complex formed by the one or more polynucleotide binding proteins and one or more loading moieties comprising a loading polynucleotide, wherein the one or more polynucleotide binding proteins are bound to the loading polynucleotide; and (b) attaching the loading polynucleotide of the one or more loading moieties of the complex to the target polynucleotide, wherein the one or more polynucleotide binding proteins of the complex retain their function and ability to bind to the loading polynucleotide of the one or more loading moieties after said attaching the loading polynucleotide of the one or more loading moieties of the complex to the target polynucleotide; and wherein the one or more polynucleotide binding proteins are derived from helicases and are stalled at one or more spacers on the loading polynucleotide.
 3. The method according to claim 1 or 2, wherein the method further comprises binding the polynucleotide binding proteins to the one or more loading moieties before step (a).
 4. The method according to claim 1 or 2, wherein the one or more polynucleotide binding proteins are two or more polynucleotide binding proteins.
 5. The method according to claim 4, wherein the two or more polynucleotide binding proteins are not directly attached to one another but are indirectly attached to one another via the one or more loading moieties.
 6. The method according to claim 4, wherein the two or more polynucleotide binding proteins are different from one another.
 7. The method according to claim 1 or 2, wherein the loading polynucleotide is a single-stranded polynucleotide.
 8. The method according to claim 1 or 2, wherein the loading polynucleotide is a Y adaptor and the Y adaptor comprises a leader sequence which preferentially threads into a pore.
 9. The method according to claim 1 or 2, wherein the loading polynucleotide is a bridging moiety, the bridging moiety is a hairpin loop and the stem of the hairpin loop is less than 200 nucleotide pairs in length. 